Endogenous l- to d-amino acid residue isomerization modulates selectivity between distinct neuropeptide receptor family members.

The l- to d-amino acid residue isomerization of neuropeptides is an understudied post-translational modification found in animals across several phyla. Despite its physiological importance, little information is available regarding the impact of endogenous peptide isomerization on receptor recognition and activation. As a result, the full roles peptide isomerization play in biology are not well understood. Here, we identify that the Aplysia allatotropin-related peptide (ATRP) signaling system utilizes l- to d-residue isomerization of one amino acid residue in the neuropeptide ligand to modulate selectivity between two distinct G protein-coupled receptors (GPCRs). We first identified a novel receptor for ATRP that is selective for the D2-ATRP form, which bears a single d-phenylalanine residue at position 2. Using cell-based receptor activation experiments, we then characterized the stereoselectivity of the two known ATRP receptors for both endogenous ATRP diastereomers, as well as for homologous toxin peptides from a carnivorous predator. We found that the ATRP system displayed dual signaling through both the Gαq and Gαs pathways, and each receptor was selectively activated by one naturally occurring ligand diastereomer over the other. Overall, our results provide insights into an unexplored mechanism by which nature regulates intercellular communication. Given the challenges in detecting l- to d-residue isomerization from complex mixtures de novo and in identifying receptors for novel neuropeptides, it is likely that other neuropeptide-receptor systems may also utilize changes in stereochemistry to modulate receptor selectivity in a manner similar to that discovered here.

Efficient biosynthesis of resveratrol via combining phenylalanine and tyrosine pathways in Saccharomyces cerevisiae.

BACKGROUND: Resveratrol is a commercially available stilbenoid widely used as dietary supplements, functional food ingredients, and cosmetic ingredients due to its diverse physiological activities. The production of resveratrol in microorganisms provides an ideal source that reduces the cost of resveratrol, but the titer in Saccharomyces cerevisiae was still much lower than that in other hosts. RESULTS: To achieve enhanced production of resveratrol in S. cerevisiae, we constructed a biosynthetic pathway via combining phenylalanine and tyrosine pathways by introducing a bi-functional phenylalanine/tyrosine ammonia lyase from Rhodotorula toruloides. The combination of phenylalanine pathway with tyrosine pathway led to a 462% improvement of resveratrol production in yeast extract peptone dextrose (YPD) medium with 4% glucose, suggesting an alternative strategy for producing p-coumaric acid-derived compounds. Then the strains were further modified by integrating multi-copy biosynthetic pathway genes, improving metabolic flux to aromatic amino acids and malonyl-CoA, and deleting by-pathway genes, which resulted in 1155.0 mg/L resveratrol in shake flasks when cultured in YPD medium. Finally, a non-auxotrophic strain was tailored for resveratrol production in minimal medium without exogenous amino acid addition, and the highest resveratrol titer (4.1 g/L) ever reported was achieved in S. cerevisiae to our knowledge. CONCLUSIONS: This study demonstrates the advantage of employing a bi-functional phenylalanine/tyrosine ammonia lyase in the biosynthetic pathway of resveratrol, suggesting an effective alternative in the production of p-coumaric acid-derived compounds. Moreover, the enhanced production of resveratrol in S. cerevisiae lays a foundation for constructing cell factories for various stilbenoids.

Expression, purification and crystallization of N-acetyl-(R)-ß-phenylalanine acylases derived from Burkholderia sp. AJ110349 and Variovorax sp. AJ110348 and structure determination of the Burkholderia enzyme.

N-Acetyl-(R)-ß-phenylalanine acylase is an enzyme that hydrolyzes the amide bond of N-acetyl-(R)-ß-phenylalanine to produce enantiopure (R)-ß-phenylalanine. In previous studies, Burkholderia sp. AJ110349 and Variovorax sp. AJ110348 were isolated as (R)-enantiomer-specific N-acetyl-(R)-ß-phenylalanine acylase-producing organisms and the properties of the native enzyme from Burkholderia sp. AJ110349 were characterized. In this study, structural analyses were carried out in order to investigate the structure-function relationships of the enzymes derived from both organisms. The recombinant N-acetyl-(R)-ß-phenylalanine acylases were crystallized by the hanging-drop vapor-diffusion method under multiple crystallization solution conditions. The crystals of the Burkholderia enzyme belonged to space group P41212, with unit-cell parameters a = b = 112.70-112.97, c = 341.50-343.32 Å, and were likely to contain two subunits in the asymmetric unit. The crystal structure was solved by the Se-SAD method, suggesting that two subunits in the asymmetric unit form a dimer. Each subunit was composed of three domains, and they showed structural similarity to the corresponding domains of the large subunit of N,N-dimethylformamidase from Paracoccus sp. strain DMF. The crystals of the Variovorax enzyme grew as twinned crystals and were not suitable for structure determination. Using size-exclusion chromatography with online static light-scattering analysis, the N-acetyl-(R)-ß-phenylalanine acylases were clarified to be dimeric in solution.

Rational design, production and in vitro analysis of photoxenoproteins.

In synthetic biology, the artificial control of proteins by light is of growing interest since it enables the spatio-temporal regulation of downstream molecular processes. This precise photocontrol can be established by the site-directed incorporation of photo-sensitive non-canonical amino acids (ncAAs) into proteins, which generates so-called photoxenoproteins. Photoxenoproteins can be engineered using ncAAs that facilitate the irreversible activation or reversible regulation of their activity upon irradiation. In this chapter, we provide a general outline of the engineering process based on the current methodological state-of-the-art to obtain artificial photocontrol in proteins using the ncAAs o-nitrobenzyl-O-tyrosine as example for photocaged ncAAs (irreversible), and phenylalanine-4'-azobenzene as example for photoswitchable ncAAs (reversible). We thereby focus on the initial design as well as the production and characterization of photoxenoproteins in vitro. Finally, we outline the analysis of photocontrol under steady-state and non-steady-state conditions using the allosteric enzyme complexes imidazole glycerol phosphate synthase and tryptophan synthase as examples.

Growth-Promotion Effects of Dissolved Amino Acids in Three Species of Hynobius Salamander Hatchlings.

It has been suggested that aquatic vertebrates may be able to meet their energy requirements by using the amino acids dissolved in environmental water. If this ability can be applied to aquatic organisms generally, then conventional ecological theories related to food web interactions should be revisited, as this would likely bring about significant advances in applications. Here, we prepared two 1 mM amino acid (phenylalanine and glycine) solutions in environmental water and conducted laboratory experiments to demonstrate the utilization of dissolved amino acids by hatchlings of three salamander species (Ezo: Hynobius retardatus, Tohoku: Hynobius lichenatus, and Japanese black: Hynobius nigrescens). Compared to controls (no amino acids in environmental water), the growth rate for Ezo salamanders was higher when larvae were reared in phenylalanine solution, while that for Japanese black salamanders was higher in glycine and phenylalanine solutions. Amino acids in environmental water had no effect on the growth of Tohoku salamanders. However, when growth was divided into early (days 1 to 5) and late (days 5 to 7) developmental stages, growth in early-developmental stage individuals was improved by phenylalanine treatment, even in Tohoku salamanders. The results showed that the growth of salamander larvae was improved when salamanders were reared in environmental water with high amino acid concentrations. Although aquatic bacteria may not have been removed completely from the environmental water, no other eukaryotes were present. Our results suggest an overlooked nutrient pathway in which aquatic vertebrates take up dissolved amino acids without mediation by other eukaryotes.

A novel L-phenylalanine dipeptide inhibits prostate cancer cell proliferation by targeting TNFSF9.

In the present study, a series of novel L-phenylalanine dipeptides were designed and synthesized by a multi-step sequence of reactions, including carbodiimide-mediated condensation, hydrolysis, mixed anhydride condensation, and nucleophilic substitution. Among them, compound 7c exhibited potent antitumor activity against prostate cancer cell PC3 in vitro and in vivo via inducing apoptosis. We investigated the significantly differentially expressed proteins in the cells caused by the compound 7c to unravel the molecular mechanisms underlying the regulation of PCa cell growth, which indicated that 7c mainly regulated the protein expression of apoptosis-related transcription factors, including c-Jun, IL6, LAMB3, OSMR, STC1, OLR1, SDC4 and PLAU; and 7c also regulated the protein expression of inflammatory cytokines including IL6, CXCL8, TNFSF9, TNFRSF12A and OSMR, and the phosphorylation levels of RelA. The action target confirmed that TNFSF9 protein is the critical binding target of 7c. These findings suggested that 7c could regulate the apoptosis and inflammatory response related signaling pathways for the inhibition of the proliferation of PC3 cells, implying that 7c could be considered a promising therapeutic candidate for PCa therapy.

Global Responses of Autopolyploid Sugarcane Badila (Saccharum officinarum L.) to Drought Stress Based on Comparative Transcriptome and Metabolome Profiling.

Sugarcane (Saccharum spp. hybrid) is frequently affected by seasonal drought, which causes substantial declines in quality and yield. To understand the drought resistance mechanisms of S. officinarum, the main species of modern sugarcane, at a molecular level, we carried out a comparative analysis of transcriptome and metabolome profiling of the sugarcane variety Badila under drought stress (DS). Compared with control group (CG) plants, plants exposed to DS had 13,744 (6663 up-regulated and 7081 down-regulated) differentially expressed genes (DEGs). GO and KEGG analysis showed that the DEGs were enriched in photosynthesis-related pathways and most DEGs had down-regulated expression. Moreover, the chlorophyll content, photosynthesis (Photo), stomatal conductance (Cond), intercellular carbon dioxide concentration (Ci) and transpiration rate (Trmmol) were sharply decreased under DS. These results indicate that DS has a significant negative influence on photosynthesis in sugarcane. Metabolome analysis identified 166 (37 down-regulated and 129 up-regulated) significantly regulated metabolites (SRMs). Over 50% of SRMs were alkaloids, amino acids and their derivatives, and lipids. The five most significantly enriched KEGG pathways among SRMs were Aminoacyl-tRNA biosynthesis, 2-Oxocarboxylic acid metabolism, Biosynthesis of amino acids, Phenylalanine metabolism, and Arginine and proline metabolism (p < 0.05). Comparing CG with DS for transcriptome and metabolome profiling (T_CG/DS and M_CG/DS, respectively), we found three of the same KEGG-enriched pathways, namely Biosynthesis of amino acids, Phenylalanine metabolism and Arginine and proline metabolism. The potential importance of Phenylalanine metabolism and Arginine and proline metabolism was further analyzed for response to DS in sugarcane. Seven SRMs (five up-regulated and two down-regulated) and 60 DEGs (17 up-regulated and 43 down-regulated) were enriched in Phenylalanine metabolism under DS, of which novel.31261, Sspon.04G0008060-1A, Sspon.04G0008060-2B and Sspon.04G0008060-3C were significantly correlated with 7 SRMs. In Arginine and proline metabolism, eight SRMs (seven up-regulated and one down-regulated) and 63 DEGs (32 up-regulated and 31 down-regulated) were enriched, of which Sspon.01G0026110-1A (OAT) and Sspon.03G0002750-3D (P5CS) were strongly associated with proline (r > 0.99). These findings present the dynamic changes and possible molecular mechanisms of Phenylalanine metabolism as well as Arginine and proline metabolism under DS and provide a foundation for future research and sugarcane improvement.

Noncanonical amino acids as doubly bio-orthogonal handles for one-pot preparation of protein multiconjugates.

Genetic encoding of noncanonical amino acid (ncAA) for site-specific protein modification has been widely applied for many biological and therapeutic applications. To efficiently prepare homogeneous protein multiconjugates, we design two encodable noncanonical amino acids (ncAAs), 4-(6-(3-azidopropyl)-s-tetrazin-3-yl) phenylalanine (pTAF) and 3-(6-(3-azidopropyl)-s-tetrazin-3-yl) phenylalanine (mTAF), containing mutually orthogonal and bioorthogonal azide and tetrazine reaction handles. Recombinant proteins and antibody fragments containing the TAFs can easily be functionalized in one-pot reactions with combinations of commercially available fluorophores, radioisotopes, PEGs, and drugs in a plug-and-play manner to afford protein dual conjugates to assess combinations of tumor diagnosis, image-guided surgery, and targeted therapy in mouse models. Furthermore, we demonstrate that simultaneously incorporating mTAF and a ketone-containing ncAA into one protein via two non-sense codons allows preparation of a site-specific protein triconjugate. Our results demonstrate that TAFs are doubly bio-orthogonal handles for efficient and scalable preparation of homogeneous protein multiconjugates.

Cognitive Functioning in Adults with Phenylketonuria in a Cohort of Spanish Patients.

The early introduction of a low phenylalanine (Phe) diet has been demonstrated to be the most successful treatment in subjects with phenylketonuria (PKU), especially for preventing severe cognitive and neurological damages. However, it still concerns that even if treated in the first months of life with supplements and following a diet, they can show slight scores below people without PKU in neuropsychological assignments. We investigated 20 adults with classical PKU aged 19-48 years (mean age 29 years) and 20 heathy controls matched by age, gender, and years of education. Patients and controls were assessed with an extended neuropsychological battery, as well as psychological aspects and quality of life, also the last Phe level result was obtained. Results showed that the most affected cognitive domains are processing speed, executive functioning, memory, and also theory of mind, but very well-preserved verbal fluency, language, and visuospatial functioning. In quality of life, some significant results were seen specially in anxiety of Phe levels, anxiety of Phe levels during pregnancy, guilt if poor adherence to supplements, and if dietary protein restriction not followed. No significant results were obtained for the psychological variables. In conclusion, it has been shown that a combination of a low Phe diet, supplement intake, and keeping Phe levels in a low range seems appropriate to have the most normal and alike cognitive performance to persons without PKU.

Entropically-Driven Co-assembly of l-Histidine and l-Phenylalanine to Form Supramolecular Materials.

Molecular self- and co-assembly allow the formation of diverse and well-defined supramolecular structures with notable physical properties. Among the associating molecules, amino acids are especially attractive due to their inherent biocompatibility and simplicity. The biologically active enantiomer of l-histidine (l-His) plays structural and functional roles in proteins but does not self-assemble to form discrete nanostructures. In order to expand the structural space to include l-His-containing materials, we explored the co-assembly of l-His with all aromatic amino acids, including phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp), all in both enantiomeric forms. In contrast to pristine l-His, the combination of this building block with all aromatic amino acids resulted in distinct morphologies including fibers, rods, and flake-like structures. Electrospray ionization mass spectrometry (ESI-MS) indicated the formation of supramolecular co-assemblies in all six combinations, but time-of-flight secondary-ion mass spectrometry (ToF-SIMS) indicated the best seamless co-assembly occurs between l-His and l-Phe while in the other cases, different degrees of phase separation could be observed. Indeed, isothermal titration calorimetry (ITC) suggested the highest affinity between l-His and l-Phe where the formation of co-assembled structures was driven by entropy. In accordance, among all the combinations, the co-assembly of l-His and l-Phe produced single crystals. The structure revealed the formation of a 3D network with nanocavities stabilized by hydrogen bonding between -N (l-His) and -NH (l-Phe). Taken together, using the co-assembly approach we expanded the field of amino acid nanomaterials and showed the ability to obtain discrete supramolecular nanostructures containing l-His based on its specific interactions with l-Phe.

Metabolomic profiling in kidney cells treated with a sodium glucose-cotransporter 2 inhibitor.

We aimed to determine the metabolomic profile of kidney cells under high glucose conditions and following sodium-glucose cotransporter 2 (SGLT2) inhibitor treatment. Targeted metabolomics using the Absolute IDQ-p180 kit was applied to quantify metabolites in kidney cells stimulated with high glucose (25 and 50 mM) and treated with SGLT2 inhibitor, dapagliflozin (2 µM). Primary cultured human tubular epithelial cells and podocytes were used to identify the metabolomic profile in high glucose conditions following dapagliflozin treatment. The levels of asparagine, PC ae C34:1, and PC ae C36:2 were elevated in tubular epithelial cells stimulated with 50 mM glucose and were significantly decreased after 2 µM dapagliflozin treatment. The level of PC aa C32:0 was significantly decreased after 50 mM glucose treatment compared with the control, and its level was significantly increased after dapagliflozin treatment in podocytes. The metabolism of glutathione, asparagine and proline was significantly changed in tubular epithelial cells under high-glucose stimulation. And the pathway analysis showed that aminoacyl-tRNA biosynthesis, arginine and proline metabolism, glutathione metabolism, valine, leucine and isoleucine biosynthesis, phenylalanine, tyrosine, and tryptophan biosynthesis, beta-alanine metabolism, phenylalanine metabolism, arginine biosynthesis, alanine, aspartate and glutamate metabolism, glycine, serine and threonine metabolism were altered in tubular epithelial cells after dapagliflozin treatment following 50 mM glucose compared to those treated with 50 mM glucose.

Metabolic phenotyping in phenylketonuria reveals disease clustering independently of metabolic control.

Phenylketonuria (PKU, MIM #261600) is one of the most common inborn errors of metabolism (IEM) with an incidence of 1:10000 in the European population. PKU is caused by autosomal recessive mutations in phenylalanine hydroxylase (PAH) and manifests with elevation of phenylalanine (Phe) in plasma and urine. Untreated PKU manifests with intellectual disability including seizures, microcephaly and behavioral abnormalities. Early treatment and good compliance result in a normal intellectual outcome in many but not in all patients. This study examined plasma metabolites in patients with PKU (n = 27), hyperphenylalaninemia (HPA, n = 1) and healthy controls (n = 32) by LC- MS/MS. We hypothesized that PKU patients would exhibit a distinct "submetabolome" compared to that of healthy controls. We further hypothesized that the submetabolome of PKU patients with good metabolic control would resemble that of healthy controls. Results from this study show: (i) Distinct clustering of healthy controls and PKU patients based on polar metabolite profiling, (ii) Increased and decreased concentrations of metabolites within and afar from the Phe pathway in treated patients, and (iii) A specific PKU-submetabolome independently of metabolic control assessed by Phe in plasma. We examined the relationship between PKU metabolic control and extended metabolite profiles in plasma. The PKU submetabolome characterized in this study represents the combined effects of dietary adherence, adjustments in metabolic pathways to compensate for defective Phe processing, as well as metabolic derangements that could not be corrected with dietary management even in patients classified as having good metabolic control. New therapeutic targets may be uncovered to approximate the PKU submetabolome to that of healthy controls and prevent long-term organ damage.

Phenylalanine conjugated supramolecular hydrogels developed from the mafenide and flurbiprofen multidrug for biological applications.

A well-known nonsteroidal anti-inflammatory drug (NSAID), flurbiprofen (FLR), was first conjugated individually with two naturally occurring amino acids such as L-phenylalanine (PHE) and L-alanine (ALA). These covalent amidic bioconjugates were further reacted individually with mafenide (a drug for treating burn wounds) and amantadine (an antiviral drug) to develop primary ammonium monocarboxylate (PAM) salts. Interestingly, both the PHE-containing multidrug salts exhibited significant gelation ability with various solvents including biologically potent water or methyl salicylate (MS). The isolated hydrogel (HG) as well as all the organogels obtained from multidrug gelators were extensively characterized by dynamic rheology and rheoreversibility studies. The hydrogel of FLR·PHE·MAF and MS gels of FLR·PHE·AMN/FLR·AMN were also selectively characterized by table-top and FEG-TEM analyses. The temperature-dependent 1H-NMR spectroscopy of the selected HG further provided insights into the gelation mechanism and the only isolated single-crystal of the weakly diffracted gelator FLR·AMN also revealed the presence of 1D hydrogen-bonded networks. The pure hydrogelator FLR·PHE·MAF salt (which is also an ambidextrous gelator) was found to be promising in both mechanical (rheoreversible) and biological applications and was found to be effective in cytotoxicity, biocompatibility, anti-cancer activity (MTT and cell migration assay), antibacterial response (zone inhibition, turbidity, INT, and resazurin assay) and haemolysis studies.

Influence of Co-amorphization on the Physical Stability and Dissolution Performance of an Anthelmintic Drug Flubendazole.

In this work, the co-amorphization approach was applied to flubendazole (FluBZ), resulting in the formation of two novel solid forms of FluBZ with l-phenylalanine (Phe) and l-tryptophan (Trp). A variety of physicochemical techniques have been used to describe new systems, including powder X-ray diffraction, thermal methods, infrared spectroscopy, and scanning electron microscopy. Co-amorphization has been shown to suppress crystallization tendency and considerably increase the shelf-life storage of amorphous flubendazole solid across a wide range of relative humidities. The dissolution behavior of the amorphous forms in biorelevant media at pH = 1.6, pH = 6.5, and 37 °C has been studied in terms of Cmax (maximum FluBZ concentration), Tmax (time to attain peak drug concentration), and AUC (concentration area under the curve during dissolution). At pH = 6.5, a continuous supersaturation and the highest AUC value of all examined systems were observed for the FluBZ-Phe (1:1) system. The phase solubility diagrams revealed that the reason for the better dissolution performance of FluBZ-Phe (1:1) at pH = 6.5 is a complexation between the components in a solution. This work highlights the applicability of co-amorphous systems in improving the physical stability and dissolution performance of drug compounds with poor biopharmaceutical characteristics.

Identification of heterochirality-mediated stereochemical interactions in peptide architectures.

In this work, we illustrate a strategy for constructing heterochiral peptide architectures with distinct structural, mechanical and thermal characteristics. A series of nanotube structures based on diphenylalanine (FF) and its chiral derivatives were examined. Pronounced effects relating to heterochirality on mechanostability and thermal stability can be identified. The homochiral peptide FF and its enantiomer ff formed nanotubes with high thermal and mechanical stabilities (Young's modulus: 20.3 ± 5.9 GPa for FF and 21.2 ± 4.7 GPa for ff). In contrast, heterochiral nanotubes formed by Ff and fF manifest superstructures along the axial direction with differed thermal and mechanical strength (Young's modulus: 7.3 ± 2.4 GPa for Ff and 8.3 ± 2.1 GPa for fF). Combining their single-crystal XRD structure and in silico results, it was demonstrated that the spatial orientations of aromatic moieties were subtly changed by heterochirality of peptide building blocks, which led to intramolecular face-to-face interactions. As the result, both intermolecular axial and interchannel interactions in heterochiral nanotubes were weakened as reflected in the strikingly deteriorated mechanical and thermal stabilities. Conversely, two aromatic side chains of the homochiral peptides were staggered and formed interdigitated steric zippers, which served as strong glues that secured the robustness of nanotubes in both axial and radial orientation. Furthermore, the generality of the heterochiral-mediated stereochemical effects was demonstrated in other "FF class" dipeptides, including fluorinated Ff, FW and FL. Our results unequivocally revealed the relationship between amino acid chirality, peptide molecule packing, and physical stabilities of "FF class" dipeptide self-assembled materials and provide valuable molecular insights into chirality-mediated stereochemical interactions in determining the properties of peptide architectures.

Effect of number of lysine motifs on the bactericidal and hemolytic activity of short cationic antimicrobial peptides.

Antimicrobial peptides (AMPs) are vital components of the nonspecific immune system that represent a promising broad-spectrum alternative to conventional antibiotics. Several short cationic antimicrobial peptides show highly effective antibacterial activity and low hemolytic activity, which are based on the action of a few critical amino acids, such as phenylalanine (F) and lysine (K). Previous studies have reported that Fmoc-based phenylalanine peptides possess appreciable antibacterial potency against Gram-positive bacteria, but their ability to kill Gram-negative bacteria was suboptimal. In this study, we designed and prepared a series of Fmoc-KnF peptide (n = 1-3) series by adding lysine motifs to strengthen their broad-spectrum antibacterial activity. The effect was investigated that the amount of lysine in Fmoc-F peptides on their antibacterial properties and hemolytic activities. Our results showed that the Fmoc-KKF peptide holds the strongest antimicrobial activity against both Gram-positive and negative bacteria among all designed peptides, as well as low hemolytic activity. These results provide support for the general strategy of enhancing the broad-spectrum antibacterial activity of AMPs through increased lysine content.

Characterization of Phenylalanine Ammonia Lyases from Lettuce (Lactuca sativa L.) as Robust Biocatalysts for the Production of d- and l-Amino Acids.

Phenylalanine ammonia lyase (PAL) catalyzes the reversible conversion of l-phenylalanine into the corresponding trans-cinnamic acid, providing a route to optically pure α-amino acids. We explored the catalytic function of all five PALs encoded in the genome of lettuce (Lactuca sativa L.) that are previously known to be involved in wound browning. All LsPALs were active toward l-phenylalanine in the ammonia elimination reaction and displayed maximum activity at 55-60 °C and pH 9.0-9.5. However, four of them, LsPAL1-LsPAL4, showed significantly higher activity and thermal stability than LsPAL5, as well as a broader substrate spectrum including some challenging substrates with steric demanding or electron-donating substituents. The best one LsPAL3 was subjected to the kinetic resolution of a panel of 21 rac-phenylalanine derivatives, as well as the ammonia addition of 21 cinnamic acid derivatives. It showed excellent enantioselectivity in most cases and significantly better activity than previously described PALs for a number of challenging non-natural substrates, demonstrating its great potential in biocatalysis.

The Utility of Amino Acid Metabolites in the Diagnosis of Major Depressive Disorder and Correlations with Depression Severity.

Major depressive disorder (MDD) is a highly prevalent and disabling condition with a high disease burden. There are currently no validated biomarkers for the diagnosis and treatment of MDD. This study assessed serum amino acid metabolite changes between MDD patients and healthy controls (HCs) and their association with disease severity and diagnostic utility. In total, 70 MDD patients and 70 HCs matched in age, gender, and ethnicity were recruited for the study. For amino acid profiling, serum samples were analysed and quantified by liquid chromatography-mass spectrometry (LC-MS). Receiver-operating characteristic (ROC) curves were used to classify putative candidate biomarkers. MDD patients had significantly higher serum levels of glutamic acid, aspartic acid and glycine but lower levels of 3-Hydroxykynurenine; glutamic acid and phenylalanine levels also correlated with depression severity. Combining these four metabolites allowed for accurate discrimination of MDD patients and HCs, with 65.7% of depressed patients and 62.9% of HCs correctly classified. Glutamic acid, aspartic acid, glycine and 3-Hydroxykynurenine may serve as potential diagnostic biomarkers, whereas glutamic acid and phenylalanine may be markers for depression severity. To elucidate the association between these indicators and clinical features, it is necessary to conduct additional studies with larger sample sizes that involve a spectrum of depressive symptomatology.

Comparison of Tandem Mass Spectrometry and the Fluorometric Method-Parallel Phenylalanine Measurement on a Large Fresh Sample Series and Implications for Newborn Screening for Phenylketonuria.

Phenylketonuria (PKU) was the first disease to be identified by the newborn screening (NBS) program. Currently, there are various methods for determining phenylalanine (Phe) values, with tandem mass spectrometry (MS/MS) being the most widely used method worldwide. We aimed to compare the MS/MS method with the fluorometric method (FM) for measuring Phe in the dried blood spot (DBS) and the efficacy of both methods in the NBS program. The FM was performed using a neonatal phenylalanine kit and a VICTOR2TM D fluorometer. The MS/MS method was performed using a NeoBaseTM 2 kit and a Waters Xevo TQD mass spectrometer. The Phe values measured with the MS/MS method were compared to those determined by the FM. The cut-off value for the NBS program was set at 120 µmol/L for FM and 85 µmol/L for MS/MS. We analyzed 54,934 DBS. The measured Phe values varied from 12 to 664 µmol/L, with a median of 46 µmol/L for the MS/MS method and from 10 to 710 µmol/L, with a median of 70 µmol/L for the FM. The Bland-Altman analysis indicated a bias of -38.9% (-23.61 µmol/L) with an SD of 21.3% (13.89 µmol/L) when comparing the MS/MS method to the FM. The Phe value exceeded the cut-off in 187 samples measured with FM and 112 samples measured with MS/MS. The FM had 181 false positives, while the MS/MS method had 106 false positives. Our study showed that the MS/MS method gives lower results compared to the FM. Despite that, none of the true positives would be missed, and the number of false-positive results would be significantly lower compared to the FM.

Combining Phenylalanine and Leucine Levels Predicts 30-Day Mortality in Critically Ill Patients Better than Traditional Risk Factors with Multicenter Validation.

In critically ill patients, risk scores are used; however, they do not provide information for nutritional intervention. This study combined the levels of phenylalanine and leucine amino acids (PLA) to improve 30-day mortality prediction in intensive care unit (ICU) patients and to see whether PLA could help interpret the nutritional phases of critical illness. We recruited 676 patients with APACHE II scores ≥ 15 or intubated due to respiratory failure in ICUs, including 537 and 139 patients in the initiation and validation (multicenter) cohorts, respectively. In the initiation cohort, phenylalanine ≥ 88.5 µM (indicating metabolic disturbance) and leucine < 68.9 µM (indicating malnutrition) were associated with higher mortality rate. Based on different levels of phenylalanine and leucine, we developed PLA scores. In different models of multivariable analyses, PLA scores predicted 30-day mortality independent of traditional risk scores (p < 0.001). PLA scores were then classified into low, intermediate, high, and very-high risk categories with observed mortality rates of 9.0%, 23.8%, 45.6%, and 81.8%, respectively. These findings were validated in the multicenter cohort. PLA scores predicted 30-day mortality better than APACHE II and NUTRIC scores and provide a basis for future studies to determine whether PLA-guided nutritional intervention improves the outcomes of patients in ICUs.