Lysine, methionine, and threonine are essential amino acids with vital functions for muscle and connective tissue health, metabolic balance, and the immune system. During illness, the demand for these amino acids typically increases, which puts patients at risk for deficiencies with harmful clinical consequences. In a secondary analysis of the Effect of Early Nutritional Support on Frailty, Functional Outcomes, and Recovery of Malnourished Medical Inpatients Trial (EFFORT), which compared individualized nutritional support to usual care nutrition in patients at nutritional risk, we investigated the prognostic impact of the lysine, methionine, and threonine metabolism. We had complete clinical and amino acid data in 237 patients, 58 of whom reached the primary endpoint of death at 30 days. In a model adjusted for comorbidities, sex, nutritional risk, and trial intervention, low plasma methionine levels were associated with 30-day mortality (adjusted HR 1.98 [95% CI 1.16 to 3.36], p = 0.01) and with a decline in functional status (adjusted OR 2.06 [95% CI 1.06 to 4.01], p = 0.03). The results for lysine and threonine did not show statistically significant differences regarding clinical outcomes. These findings suggest that low levels of methionine may be critical during hospitalization among patients at nutritional risk. Further studies should investigate the effect of supplementation of methionine in this patient group to improve outcomes.

The neuronal microtubule-associated tau protein is characterized in vivo by a large number of post-translational modifications along the entire primary sequence that modulates its function. The primary modification of tau is phosphorylation of serine/threonine or tyrosine residues that is involved in the regulation of microtubule binding and polymerization. In neurodegenerative disorders referred to as tauopathies including Alzheimer\'s disease, tau is abnormally hyperphosphorylated and forms fibrillar inclusions in neurons progressing throughout different brain area during the course of the disease. The O-β-linked N-acetylglucosamine (O-GlcNAc) is another reversible post-translational modification of serine/threonine residues that is installed and removed by the unique O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA), respectively. This modification was described as a potential modulator of tau phosphorylation and functions in the physiopathology. Moreover, reducing protein O-GlcNAc levels in the brain upon treatment of tauopathy mouse models with an OGA inhibitor reveals a beneficial effect on tau pathology and neurodegeneration. However, whether the role of tau O-GlcNAcylation is responsible of the protective effect against tau toxicity remains to be determined. The production of O-GlcNAc modified recombinant tau protein is a valuable tool for the investigations of the impact of O-GlcNAcylation on tau functions, modulation of interactions with partners and crosstalk with other post-translational modifications, including but not restricted to phosphorylation. We describe here the in vitro O-GlcNAcylation of tau with recombinant OGT for which we provide an expression and purification protocol. The use of the O-GlcNAc tau protein in functional studies requires the analytical characterization of the O-GlcNAc pattern. Here, we describe a method for the O-GlcNAc modification of tau protein with recombinant OGT and the analytical characterization of the resulting O-GlcNAc pattern by a combination of methods for the overall characterization of tau O-GlcNAcylation by chemoenzymatic labeling and mass spectrometry, as well as the quantitative, site-specific pattern by NMR spectroscopy.

Bursaphelenchus xylophilus, the pine wood nematode (PWN), is the causal agent of pine wilt disease (PWD), which causes enormous economic loss annually. According to our previous research, fomepizole, as a selective inhibitor of PWN alcohol dehydrogenase (ADH), has the potential to be a preferable lead compound for developing novel nematicides. However, the underlying molecular mechanism is still unclear. The result of molecular docking showed that the stronger interactions between fomepizole and PWN ADH at the active site of ADH were attributed to hydrogen bonds. Low-dose fomepizole had a substantial negative impact on the egg hatchability, development, oviposition, and lifespan of PWN. Transcriptome analysis indicated that 2,124 upregulated genes and 490 downregulated genes in fomepizole-treated PWN were obtained. Kyoto Encyclopedia of Genes and Genomes enrichment analysis of differentially expressed genes indicated that fomepizole could be involved in controlling PWN vitality mainly by regulating key signaling pathways, such as the ribosome, hippo signaling pathway, and lysosome. Remarkably, the results of RNA interference indicated that the downregulated serine/threonine-protein phosphatase gene (stpp) could reduce the egg hatchability, development, oviposition, and lifespan of PWN, which was closely similar to the consequences of nematodes with low-dose fomepizole treatment. In addition, the silencing of stpp resulted in weakness of PWN pathogenicity, which indicated that stpp could be a potential drug target to control PWN.

BACKGROUND: Targeting receptor-interacting serine/threonine protein kinase 1 could mitigate the devastating sequelae of the hyperinflammatory state observed in severe cases of COVID-19. This study explored the immunomodulatory and clinical effects of the receptor-interacting serine/threonine protein kinase 1 inhibitor SAR443122 (eclitasertib) in patients with severe COVID-19.
METHODS: In this Phase 1b, double-blinded, placebo-controlled study (NCT04469621) a total of 82 patients were screened, of whom 68 patients were eligible and randomized (2:1) to receive eclitasertib 600 mg (300 mg twice daily) or placebo up to 14 days. Primary outcome was relative change in C-reactive protein from baseline to Day 7. Time to clinical improvement using 7-point ordinal scale, ventilator/respiratory failure-free days, change in SpO2/FiO2 ratio, and biomarkers of severe COVID-19 were explored.
RESULTS: Geometric mean ratio (point estimate [90% confidence interval]) of the relative change from baseline in C-reactive protein with eclitasertib vs. placebo on Day 7 was 0.85 (0.49-1.45; p = 0.30). Median time to 50% decrease in C-reactive protein from baseline was 3 days vs. 5 days (p = 0.056) with eclitasertib vs. placebo. Median time to ≥ 2-point improvement on 7-point clinical symptoms scale was 8 days vs. 10 days with eclitasertib vs. placebo (p = 0.38). Mean ventilator/respiratory failure-free days, change in baseline-adjusted SpO2/FiO2 ratio, and clinical biomarkers showed consistent numerical improvements with eclitasertib vs. placebo. The most frequently reported treatment-emergent adverse events were gastrointestinal disorders and condition aggravated/worsened COVID-19 pneumonia.
CONCLUSIONS: Eclitasertib was well tolerated with consistent trends toward more rapid resolution of inflammatory biomarkers and clinical improvement in severe COVID-19 patients than placebo.
RESULTS:
UNASSIGNED: NCT04469621, first posted on clinicaltrials.gov on July 14, 2020.

Therapeutic monoclonal antibodies are the most rapidly growing class of molecular medicine, and they are beneficial to the treatment of a broad spectrum of human diseases. However, the aggregation of antibodies during the process of manufacture, distribution, and storage poses significant challenges, potentially compromising efficacy and inducing adverse immune responses. We previously conceived a QTY (glutamine, threonine, tyrosine) code, a simple tool for enhancing protein water-solubility by systematically pairwise replacing hydrophobic residues L (leucine), V (valine)/I (isoleucine), and F (phenylalanine). The QTY code offers a promising alternative to traditional methods of controlling aggregation in integral transmembrane proteins. In this study, we designed variants of four antibodies applying the QTY code, changing only the β-sheets. Through the structure-based aggregation analysis, we found that these QTY antibody variants demonstrated significantly decreased aggregation propensity compared to their wild-type counter parts. Our results of molecular dynamics simulations showed that the design by QTY code is capable of maintaining the antigen-binding affinity and structural stability. Our structural informatic and computational study suggests that the QTY code offers a significant potential in mitigating antibody aggregation.

This study aims to explore the relationship between macrosomia and amino acids in maternal and cord sera.
METHODS: In the case-control study, 78 pairs of mothers and newborns were recruited from December 2016 to November 2019. Participants were divided into the macrosomia group (BW ≥ 4000 g, n = 39) and the control group (BW between 2500 g and 3999 g, n = 39) according to the birth weight (BW) of newborns. Maternal vein blood samples were collected before delivery and cord vein blood samples were collected after birth. The levels of amino acids in maternal and cord sera were measured by liquid chromatography and mass spectrometry (LC-MS/MS) in the year 2021. The difference in amino acid levels in maternal and cord sera between the two groups was compared, and the contribution of each amino acid to the difference between the two groups was analyzed. Unconditional logistic regression analysis was used to test the relationship between macrosomia and amino acids.
RESULTS: In maternal serum during the antepartum, the levels of asparagine, glutamine, methionine, alanine, and threonine in the macrosomia group were higher but arginine was lower than that in the control group (p < 0.05). In cord serum, the levels of lysine, histidine, phenylalanine, arginine, tryptophan, valine, isoleucine, glutamate, tyrosine, and total essential amino acid (EAA) in the macrosomia group were lower while glutamine was higher than that in the control group (p < 0.05). The ratios of EAA, valine, threonine, methionine, tryptophan, and alanine in maternal serum to those in cord serum were higher, while the ratio of glutamine was lower in the macrosomia group (p < 0.05). Arginine and threonine in maternal serum and glutamate, glutamine, and histidine in cord serum were associated with macrosomia (p < 0.05).
CONCLUSIONS: Most of the amino acid levels in the maternal sera of the macrosomia group are higher than those in the control group, while most of the amino acids\' levels in the cord sera of the macrosomia group are lower than those in the control group. The ratios of some amino acids in maternal serum to those in cord serum were different between the two groups. Arginine and threonine in maternal serum and glutamate, glutamine, and histidine in cord serum are closely related to macrosomia.

OBJECTIVE: Enhancing iron absorption and utilization is important for amelioration iron status faster and thereby, for improving quality of life. Dietary protein and amino acids, including methionine and threonine, have been reported to facilitate the absorption and utilization of dietary iron. Here, we investigated the effect of combined ingestion of methionine, threonine, and iron on the improvement of iron status during a short-term intervention, by comparing that with iron ingestion alone in healthy young women.
METHODS: This was a randomized, double-blind, parallel-group, comparative study with 45 participants (aged 20-39) randomly assigned to three groups (n = 15 each): one group was administered 200 mg methionine, 400 mg threonine, and 6 mg iron once daily (FEMT); another ingested 6 mg iron alone (FE); and the third group ingested a placebo (PCG). Blood samples and dietary nutrient data were collected before the intervention (week 0) and after 2, 4, and 6 weeks. Serum iron, hemoglobin, transferrin, and ferritin levels were measured.
RESULTS: Blood hemoglobin levels were significantly higher in the FEMT than in the FE group (P < 0.05) at week 4. Serum iron, transferrin, and ferritin levels were not changed across groups. In addition, our analyses showed that the observed increase in hemoglobin levels was affected by the intervention rather than changes in dietary nutrient intake.
CONCLUSIONS: Ingestion of methionine and threonine with low doses of iron leads to a higher hemoglobin levels than that with iron alone in a short period of 4 weeks.
BACKGROUND: University Hospital Medical Information Network Clinical Trial Registry (UMIN000046621).

Menopausal syndrome (MS) refers to a series of symptoms with autonomic nervous system dysfunction caused by decreased sex hormones before and after menopause. Baihe Dihuang (BHDH) decoction positively affects MS, but its mechanism remains unclear. This study aimed to reveal the underlying mechanism through network pharmacology. The components of the BHDH Decoction were found through HERB, while corresponding targets were obtained from the HERB, Drug Bank, NPASS, Targetnet, and Swisstarget databases. The MS targets were obtained from GeneCards and OMIM. STRING was used to construct the protein-protein interaction networks. OmicShare tools were used for Gene Ontology and Kyoto encyclopedia of genes and genomes analyses. Finally, Autodock Vina 1.1.2 software (https://vina.scripps.edu/downloads/) was used for molecular alignment to verify whether the main active ingredients and key targets had good binding activity. We screened out 27 active ingredients and 251 effective targets of BHDH Decoction, 3405 MS-related targets, and 133 intersection targets between BHDH Decoction and MS. Protein-protein interaction network identified tumor protein P53, Serine/threonine-protein kinase AKT, epidermal growth factor receptor, Estrogen Receptor 1, and jun proto-oncogene as critical targets. Gene ontology analysis showed that these targets were mainly involved in the cellular response to chemical stimulus, response to oxygen-containing compound, cellular response to endogenous stimulus, response to an organic substance, and response to chemical, etc. Kyoto encyclopedia of genes and genomes pathways were mainly enriched in endocrine resistance, pathways in cancer, and the ErbB signaling pathway, etc. Molecular docking results showed that emodin and stigmasterol are strongly associated with Serine/threonine-protein kinase AKT, Estrogen Receptor 1, epidermal growth factor receptor, sarcoma gene, and tumor protein P53. This study preliminarily revealed the multi-component, multi-target, and multi-channel mechanism of BHDH Decoction in treating MS. It provides a reference for in vitro and in vivo research and clinical application of BHDH Decoction in the treatment of MS.

Dietary supplementation with methionine and threonine spares body protein in rats fed a low protein diet, but the effect is not observed for other essential amino acids. Although the requirement for sulfur amino acids is relatively high in rodents, the precise mechanisms underlying protein retention are not fully understood. The aim of this study was to explore whether the activation of mammalian target of rapamycin complex 1 (mTORC1) downstream factors in skeletal muscle by supplementation with threonine and/or methionine contributes to protein retention under sufficient cystine requirement. Male Sprague-Dawley rats were freely fed a 0% protein diet for 2 weeks. These experimental rats were then fed a restricted diet (14.5 g/day) containing 12% soy protein supplemented with both cystine and, methionine and threonine (MT), methionine (M), threonine (T), or neither (NA) (n = 8) for an additional 12 days. Two additional groups were freely fed a diet containing 0% protein or 20% casein as controls (n = 6). Body weight and gastrocnemius muscle weight were higher, and blood urea nitrogen and urinary nitrogen excretion were lower, in the M and MT groups than in the T and NA groups, respectively. p70 S6 kinase 1 abundance was higher, and eukaryotic translation initiation factor 4E-binding protein 1 abundance and mRNA levels were lower, in the skeletal muscles of the M and MT groups. These results suggest that methionine regulates mTORC1 downstream factors in skeletal muscle, leading to spare body protein in rats fed a low protein diet meeting cystine requirements.

The burden of diabetic retinopathy (DR) is increasing, and the sensitive biomarkers of the disease were not enough. Studies have found that the metabolic profile, such as amino acid (AA) and acylcarnitine (AcylCN), in the early stages of DR patients might have changed, indicating the potential of metabolites to become new biomarkers. We are amid to construct a metabolite-based prediction model for DR risk. This study was conducted on type 2 diabetes (T2D) patients with or without DR. Logistic regression and extreme gradient boosting (XGBoost) prediction models were constructed using the traditional clinical features and the screening features, respectively. Assessing the predictive power of the models in terms of both discrimination and calibration, the optimal model was interpreted using the Shapley Additive exPlanations (SHAP) to quantify the effect of features on prediction. Finally, the XGBoost model incorporating AA and AcylCN variables had the best comprehensive evaluation (ROCAUC = 0.82, PRAUC = 0.44, Brier score = 0.09). C18 : 1OH lower than 0.04 μmol/L, C18 : 1 lower than 0.70 μmol/L, threonine higher than 27.0 μmol/L, and tyrosine lower than 36.0 μmol/L were associated with an increased risk of developing DR. Phenylalanine higher than 52.0 μmol/L was associated with a decreased risk of developing DR. In conclusion, our study mainly used AAs and AcylCNs to construct an interpretable XGBoost model to predict the risk of developing DR in T2D patients which is beneficial in identifying high-risk groups and preventing or delaying the onset of DR. In addition, our study proposed possible risk cut-off values for DR of C18 : 1OH, C18 : 1, threonine, tyrosine, and phenylalanine.