Alterations in the Gut Microbiota and Metabolomics of Seafarers after a Six-Month Sea Voyage.

Maintaining the health of seafarers is a difficult task during long-term voyages. Little is known about the corresponding changes in the gut microbiome-host interaction. This study recruited 30 seafarers undertaking a 6-month voyage and analyzed their gut microbiota using 16S rRNA gene sequencing. Fecal untargeted metabolomics analysis was performed using liquid chromatography-mass spectrometry. Significant changes in the composition of the gut microbiota and an increased ratio of Firmicutes/Bacteroidetes at the end (day 180) of the 6-month voyage, relative to the start (day 0), were observed. At the genus level, the abundances of Holdemanella and Plesiomonas were significantly increased, while the abundance of Bacteroides was decreased. Predicted microbial functional analysis revealed significant decreases in folate biosynthesis and biotin metabolism. Furthermore, 20 differential metabolites within six differentially enriched human metabolic pathways (including arginine biosynthesis, lysine degradation, phenylalanine metabolism, sphingolipid metabolism, pentose and glucuronate interconversions, and glycine, serine, and threonine metabolism) were identified by comparing the fecal metabolites at day 0 and day 180. Spearman correlation analysis revealed close relationships between the 14 differential microbiota members and the six differential fecal metabolites that might affect specific human metabolic pathways. This study adopted a multi-omics approach and provides potential targets for maintaining the health of seafarers during long-term voyages. These findings are worthy of more in-depth exploration in future studies. IMPORTANCE Maintaining the health of seafarers undertaking long-term voyages is a difficult task. Apart from the alterations in the gut microbiome and fecal metabolites after a long-term voyage, our study also revealed that 20 differential metabolites within six differentially enriched human metabolic pathways are worthy of attention. Moreover, we found close relationships between the 14 differential microbiota members and the six differential fecal metabolites that might impact specific human metabolic pathways. Accordingly, preventative measures, such as adjusting the gut microbiota by decreasing potential pathobionts or increasing potential probiotics as well as offsetting the decrease in B vitamins and beneficial metabolites (e.g., d-glucuronic acid and citrulline) via dietary adjustment or nutritional supplements, might improve the health of seafarers during long-term sea voyages. These findings provide valuable clues about gut microbiome-host interactions and propose potential targets for maintaining the health of seafarers engaged in long-term sea voyages.

Quantitative analysis of γ-glutamylisoleucine, γ-glutamylthreonine, and γ-glutamylvaline in HeLa cells using UHPLC-MS/MS.

γ-Glutamylpeptides have been identified as potential biomarkers for a number of diseases including cancer, diabetes, and liver disease. In this study, we developed and validated a novel quantitative analytical strategy for measuring γ-glutamylisoleucine, γ-glutamylthreonine, and γ-glutamylvaline, all of which have been previously reported as potential biomarkers for prostate cancer in HeLa cells using ultra-high-performance liquid chromatography-tandem mass spectrometry. A BEH C18 column was used as the stationary phase. Mobile phase A was 99:1 water:formic acid and mobile phase B was acetonitrile. Chemical isotope labeling using benzoyl chloride was used as the internal standardization strategy. Sample preparation consisted of the addition of water to a frozen cell pellet, sonication, derivatization, centrifugation, and subsequent addition of an internal standard solution. The method was validated for selectivity, accuracy, precision, linearity, and stability. The determined concentrations of γ-glutamylisoleucine, γ-glutamylthreonine, and γ-glutamylvaline in HeLa cells were 1.92 ± 0.06, 10.8 ± 0.4, and 1.96 ± 0.04 pmol/mg protein, respectively. In addition, the qualitative analysis of these analytes in human serum was achieved using a modified sample preparation strategy. To the best of our knowledge, this is the first report of the use of benzoyl chloride for chemical isotope labeling for metabolite quantitation in cells.

Spectral editing of alanine, serine, and threonine in uniformly labeled proteins based on frequency-selective homonuclear recoupling in solid-state NMR.

Spectral editing is crucial to simplify the crowded solid-state NMR spectra of proteins. New techniques are introduced to edit 13C-13C correlations of uniformly labeled proteins under moderate magic-angle spinning (MAS), based on our recent frequency-selective homonuclear recoupling sequences [Zhang et al., J. Phys. Chem. Lett. 2020, 11, 8077-8083]. The signals of alanine, serine, or threonine residues are selected out by selective 13Cα-13Cß double-quantum filtering (DQF). The 13Cα-13Cß correlations of alanine residues are selectively established with efficiency up to ~ 1.8 times that by dipolar-assisted rotational resonance (DARR). The techniques are shown in 2D/3D NCCX experiments and applied to the uniformly 13C, 15N labeled Aquaporin Z (AqpZ) membrane protein, demonstrating their potential to simplify spectral analyses in biological solid-state NMR.

Simple and Rapid (Extraction) Protocol for NMR Metabolomics-Direct Measurement of Hydrophilic and Hydrophobic Metabolites Using Slice Selection.

Investigating the metabolic profiles of solid sample materials with solution nuclear magnetic resonance (NMR) spectroscopy requires the extraction of these metabolites. This is commonly done by using two immiscible solvents such as water and chloroform for extraction. Subsequent solvent removal makes these extraction procedures very time-consuming. To shorten the preparation time of the NMR sample, the following protocol is proposed: the metabolites from a solid or liquid sample are extracted directly in the NMR tube, the NMR tube is centrifuged, and the metabolite profiles in the aqueous and organic phases are determined by using slice-selective proton NMR experiments. This protocol was tested with 11 black teas and 11 green teas, which can be easily distinguished by their metabolic profiles in the aqueous phase. As a test case for liquid samples, 29 milk samples were investigated. The geographical origin of the diaries where the milk was processed could not be determined unequivocally from the metabolic profiles of the hydrophilic metabolites; however, this was easily seen in the lipid profiles. As shown for the different test samples, the extraction protocol in combination with slice-selection NMR experiments is suitable for metabolic investigations. Because samples are rapidly processed, this approach can be used to explore different extraction strategies for metabolite isolation.

Enhanced O-succinyl-l-homoserine production by recombinant Escherichia coli ΔIJBB*TrcmetL/pTrc-metAfbr -Trc-thrAfbr -yjeH via multilevel fermentation optimization.

AIMS: Constructing a strain with high yield of O-succinyl-l-homoserine (OSH) and improving the titre through multilevel fermentation optimization. METHODS AND RESULTS: OSH high-yielding strain was first constructed by deleting the thrB gene to block the threonine biosynthesis. Single-factor experiment was carried out, where a Plackett-Burman design was used to screen out three factors (glucose, yeast and threonine) from the original 11 factors that affected the titre of OSH. The Box-Behnken response surface method was used to optimize the fermentation conditions. Through gene editing and medium optimization, the titre of OSH increased from 7·20 to 8·70 g l-1 in 500 ml flask. Furthermore, the fermentation process and fed-batch fermentation conditions including pH, temperature, feeding strategy and feeding medium were investigated and optimized. Under the optimal conditions, the titre of OSH reached 102·5 g l-1 , which is 5·6 times higher than before (15·6 g l-1 ). CONCLUSIONS: O-succinyl-l-homoserine fermentation process was established and the combination of response surface methodology and metabolic pathway analysis effectively improved the titre of OSH. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, the titre of OSH reached the needs for industrial production and the metabolic pathway of OSH was demonstrated for further optimization.

Variation in lysine, threonine, and tryptophan availability in meat and bone meal as estimated by the slope-ratio growth assay technique in growing rats.

Meat and bone meal (MBM) is an important protein source used in animal feeds. However, as the composition and availability of amino acids (AAs) in MBM fluctuate markedly, it is important to verify the magnitude of these parameters in different MBMs. In this study, the AA compositions of 19 MBMs were analyzed to confirm variations in lysine (Lys), threonine (Thr), and tryptophan (Trp), then which were compared with those of soybean meal (SBM) and fish meal (FM). Instability in Lys, Thr, and Trp availabilities in six MBMs were also considered after estimation using the slope-ratio growth assay technique in rats. Variations in AA composition were evaluated using the coefficient of variance (CV: Standard deviation/Mean). CVs for Lys, Thr, and Trp content were 9.40, 11.83, and 18.12 in MBM, 2.71, 2.48, and 3.19 in SBM, and 10.09, 10.44, and 13.47 in FM. Furthermore, means and SDs for Lys, Thr, and Trp availabilities in MBM were 53.3 ± 10.4% (CV: 19.5), 65.9 ± 17.6% (CV: 26.6), and 83.2 ± 11.2% (CV: 13.5), respectively. These results provide the first evidence that variations in MBM AA compositions were 3.5 to 5.7 times higher than those in SBM, but similar to FM, and that the large variation in availability substantially existed.

Selective targeting of signet ring cell adenocarcinomas.

Many epithelial tumors, especially signet-ring cell adenocarcinomas, produce huge amounts of mucin glycoproteins that fill cytoplasm and push nucleus to the periphery, giving a signet ring like structure to the cell. Mucin proteins are very rich of l-threonine which is essential in humans. L-threonine content can reach up to 35% of total amino acid composition of some mucin proteins. Therefore l-threonine can be the Achilles heel of signet ring cell adenocarcinomas which are one of the most malignant and agressive cancers. A modified bioisoster of l-threonine, 4-fluoro l-threonine (its fluorine can be radioactive or not), can be used to selectively kill signet ring cancer cells without harming normal cells or for diagnostic purposes.

Method for Identification of Threonine Isoforms in Peptides by Ultraviolet Photofragmentation of Cold Ions.

Identification of isomeric amino acid residues in peptides and proteins is challenging but often highly desired in proteomics. One of the practically important cases that require isomeric assignments is that associated with single-nucleotide polymorphism substitutions of Met residues by Thr in cancer-related proteins. These genetically encoded substitutions can yet be confused with the chemical modifications, arising from protein alkylation by iodoacetamide, which is commonly used in the standard procedure of sample preparation for proteomic analysis. Similar to the genetically encoded mutations, the alkylation also induces a conversion of methionine residues, but to the iso-threonine form. Recognition of the mutations therefore requires isoform-sensitive detection techniques. Herein, we demonstrate an analytical method for reliable identification of isoforms of threonine residues in tryptic peptides. It is based on ultraviolet photodissociation mass spectrometry of cryogenically cooled ions and a machine-learning algorithm. The measured photodissociation mass spectra exhibit isoform-specific patterns, which are independent of the residues adjacent to threonine or iso-threonine in a peptide sequence. A comprehensive metric-based evaluation demonstrates that, being calibrated with a set of model peptides, the method allows for isomeric identification of threonine residues in peptides of arbitrary sequence.

Evaluation of the optimal standardized ileal digestible threonine:lysine ratio in lactating sow diets.

Camborough PIC sows (n = 291) were fed 1 of 5 dietary treatments to evaluate the standardized ileal digestible (SID) Thr:Lys requirement during lactation. Sows were blocked by parity (1, 2, and 3+) and randomly allotted to 1 of 5 SID Thr:Lys ratios (52, 60, 68, 76, and 84). SID Lys was formulated to 1.03% and SID methionine and cystine (M + C):Lys was 55%. Sows were allowed 5.45 kg feed/day starting on the day of farrowing. All other nutrients met or exceeded the NRC (2012) requirements. Sows were allowed ad libitum access to water, and piglets were cross-fostered within treatment within 24 h of birth. Data collected during the study included sow prefarrow BW, sow weaning weight, starting litter weight, weaning litter weight, piglet mortality, weaning-to-estrus interval, and subsequent total born piglets. Data were analyzed as a randomized complete block design using PROC MIXED with sow as the experimental unit and treatment and parity as fixed effects. Requirement estimation models evaluated were quadratic polynomial, broken-line linear, and broken-line quadratic (BLQ) using PROC NLMIXED. Models that differed in their Bayesian information criterion (BIC) values by at least 2 points were considered to have meaningful differences in their data fit. The best-fit model was based on the best BIC value. As the SID Thr:Lys ratio increased, there was a quadratic response on ADFI (4.73, 4.57, 4.67, 4.55, and 4.60 kg/d; P = 0.001) and percent of sow BW loss (-6.4, -7.89, -7.35, -6.19, and -5.81; P = 0.004). In addition, there was a trend for weaning-to-estrus interval to be affected by the ratio (5.4, 4.4, 4.6, 4.5, and 4.8; P = 0.07). Daily litter gain was also quadratically improved as the SID Thr:Lys ratio increased (2.67, 2.67, 2.78, 2.77, and 2.68 kg/d; P-value = 0.001). For daily litter gain, the BLQ model was determined to be the best fit with an estimated SID Thr:Lys requirement of 65%. In conclusion, the optimal SID Thr:Lys ratio for litter growth was 65.

Hydrazide functionalized magnetic nanoparticles for specific extraction of N-terminal serine and threonine peptides.

In this study, we present hydrazide functionalized magnetic nanoparticles as a sorbent prepared by a new and facile method. Scanning electron microscope and Fourier transform infrared were used for characterizing the synthesized nanoparticles. The ability of the sorbent to extract N-terminal serine and threonine peptides was evaluated. The peptides were modified by oxidation of the hydroxyl group in the 1,2-amino alcohol structure before extraction. These aldehyde-forms of peptides were specifically bonded to the hydrazide groups of the sorbent. The formed hydrazone bonds were cleaved in the presence of hydroxylamine reagent. Finally, the oximated peptides were released and quantified with a high-performance liquid chromatography-diode array spectroscopy. The effects of experimental parameters including extraction time, elution time and elution volume on extraction efficiency were also investigated. The required time for the extraction process to reach equilibrium and elution time was only 8 h. The adsorption efficiency of the sorbent was 79 and 77% for peptides with N-terminal serine and threonine, respectively. The sorbent showed good specificity for extracting the peptides. In addition, the extraction efficiency of the sorbent remained constant in the presence of a non-N-terminal serine and threonine peptide as interference.

Threonine 454 phosphorylation in Grainyhead-like 3 is important for its function and regulation by the p38 MAPK pathway.

The mammalian Grainyhead-like 3 (GRHL3) transcription factor is essential for epithelial development and plays a protective role against squamous cell carcinoma of the skin and of the oral cavity. A single nucleotide polymorphism (SNP) in GRHL3, rs141193530 (p.P455A), is associated with non-melanoma skin cancer in human patients. Moreover, it is known that this SNP, as well as another variant, rs41268753 (p.T454M), are associated with nonsyndromic cleft palate and that rs41268753 negatively affects GRHL3 transcriptional activity. These SNPs are located in adjacent codons of the GRHL3 gene, and the occurrence of either SNP abolishes a putative threonine-proline phosphorylation motif at T454 in the encoded protein. The role of phosphorylation in regulating mammalian GRHL function is currently unknown. In this work we show that GRHL3 is phosphorylated at several residues in a human keratinocyte cell line, among them at T454. This site is essential for the full transcriptional activity of GRHL3. The T454 residue is phosphorylated by p38 MAPK in vitro and activation of p38 signaling in cells causes an increase in GRHL3 activity. The regulation of GRHL3 function by this pathway is dependent on T454, as the substitution of T454 with methionine inhibits the activation of GRHL3. Taken together, our results show that T454 is one of the phosphorylated residues in GRHL3 in keratinocytes and this residue is important for the upregulation of GRHL3 transcriptional activity by the p38 pathway.

Varying the ratio of Lys:Met while maintaining the ratios of Thr:Phe, Lys:Thr, Lys:His, and Lys:Val alters mammary cellular metabolites, mammalian target of rapamycin signaling, and gene transcription.

Amino acids are not only precursors for but also signaling molecules regulating protein synthesis. Regulation of protein synthesis via AA occurs at least in part by alterations in the phosphorylation status of mammalian target of rapamycin (mTOR) pathway proteins. Although the ideal profile of Lys:Met to promote milk protein synthesis during established lactation in dairy cows has been proposed to be 3:1, aside from being the most-limiting AA for milk protein synthesis, the role of Met in other key biologic pathways such as methylation is not well characterized in the bovine. The objective of this study was to determine the influence of increasing supplemental Met, based on the ideal 3:1 ratio of Lys to Met, on intracellular metabolism related to protein synthesis and mTOR pathway phosphorylation status. MAC-T cells, an immortalized bovine mammary epithelial cell line, were incubated (n = 5 replicates/treatment) for 12 h with 3 incremental doses of Met while holding Lys concentration constant to achieve the following: Lys:Met 2.9:1 (ideal AA ratio; IPAA), Lys:Met 2.5:1 (LM2.5), and Lys:Met 2.0:1 (LM2.0). The ratios of Thr:Phe (1.05:1), Lys:Thr (1.8:1), Lys:His (2.38:1), and Lys:Val (1.23:1) were the same across the 3 treatments. Applying gas chromatography-mass spectrometry metabolomics revealed distinct clusters of differentially concentrated metabolites in response to Lys:Met. Lower Phe, branched-chain AA, and putrescine concentrations were observed with LM2.5 compared with IPAA. Apart from greater intracellular Met concentrations, further elevations in Met level (LM2.0) led to greater intracellular concentrations of nonessential AA (Pro, Glu, Gln, and Gly) compared with IPAA and greater essential AA (EAA; Met, Ile, and Leu) and nonessential AA (Pro, Gly, Ala, Gln, and Glu) compared with LM2.5. However, compared with IPAA, mRNA expression of ß-casein and AA transporters (SLC7A5, SLC36A1, SLC38A2, SLC38A9, and SLC43A1) and mTOR phosphorylation were lower in response to LM2.5 and LM2.0. Overall, the results of this study provide evidence that increasing Met while Lys and the ratios of Phe, Thr, His, and Val relative to Lys were held constant could increase the concentration and utilization of intracellular EAA, in particular branched-chain AA, potentially through improving the activity of AA transporters partly controlled by mTOR signaling. Because EAA likely are metabolized by other tissues upon absorption, a question for future in vivo studies is whether formulating diets for optimal ratios of EAA in the metabolizable protein is sufficient to provide the desired levels of these AA to the mammary cells.

O-GlcNAcylation of amyloid-ß precursor protein at threonine 576 residue regulates trafficking and processing.

The pathological hallmark of Alzheimer's disease (AD) is associated with the accumulation of amyloid-ß (Aß) derived from proteolytic processing of amyloid-ß precursor protein (APP). APP undergoes post-translational modification including N- and O-glycosylation. O-GlcNAcylation is a novel type of O-glycosylation, mediated by O-GlcNAc transferase attaching O-ß-N-acetylglucosamine (O-GlcNAc) to serine/threonine residues of the target proteins. O-GlcNAc is removed by O-GlcNAcase. We have previously reported that increasing O-GlcNAcylated APP using the O-GlcNAcase inhibitor, PUGNAc, increases its trafficking rate to the plasma membrane and decreases its endocytosis rate, resulting in decreased Aß production. However, O-GlcNAc modification sites in APP are unknown. In this study, we mutated three predicted O-GlcNAc modification threonine residues of APP into alanines (T291A, T292A, and T576A) and expressed them in HeLa cells. These APP mutants showed reduced O-GlcNAcylation levels, indicating that these sites were endogenously O-GlcNAcylated. Thr 576 was the major O-GlcNAcylation site when cell was treated with PUGNAc. We also showed that the effects of PUGNAc on APP trafficking to the plasma membrane and Aß production were prevented in the T576A mutant. These results implicate Thr 576 as the major O-GlcNAcylation site in APP and indicate that O-GlcNAcylation of this residue regulates its trafficking and processing. Thus, specific O-GlcNAcylation of APP at Thr 576 may be a novel and promising drug target for AD therapeutics.

Development of a Liquid Chromatography-High Resolution Mass Spectrometry Metabolomics Method with High Specificity for Metabolite Identification Using All Ion Fragmentation Acquisition.

High-resolution mass spectrometry (HRMS)-based metabolomics approaches have made significant advances. However, metabolite identification is still a major challenge with significant bottleneck in translating metabolomics data into biological context. In the current study, a liquid chromatography (LC)-HRMS metabolomics method was developed using an all ion fragmentation (AIF) acquisition approach. To increase the specificity in metabolite annotation, four criteria were considered: (i) accurate mass (AM), (ii) retention time (RT), (iii) MS/MS spectrum, and (iv) product/precursor ion intensity ratios. We constructed an in-house mass spectral library of 408 metabolites containing AMRT and MS/MS spectra information at four collision energies. The percent relative standard deviations between ion ratios of a metabolite in an analytical standard vs sample matrix were used as an additional metric for establishing metabolite identity. A data processing method for targeted metabolite screening was then created, merging m/z, RT, MS/MS, and ion ratio information for each of the 413 metabolites. In the data processing method, the precursor ion and product ion were considered as the quantifier and qualifier ion, respectively. We also included a scheme to distinguish coeluting isobaric compounds by selecting a specific product ion as the quantifier ion instead of the precursor ion. An advantage of the current AIF approach is the concurrent collection of full scan data, enabling identification of metabolites not included in the database. Our data acquisition strategy enables a simultaneous mixture of database-dependent targeted and nontargeted metabolomics in combination with improved accuracy in metabolite identification, increasing the quality of the biological information acquired in a metabolomics experiment.

Effect of a low crude protein diet supplemented with different levels of threonine on growth performance, carcass traits, blood parameters, and immune responses of growing broilers.

A study was conducted to evaluate growth performance, carcass traits, blood serum parameters, and immune responses of Ross 308 male broilers fed diets containing 2 different crude protein (CP) levels (97.5 and 100%) and 4 threonine (Thr) levels (100, 110, 120, and 130% of Ross recommendations for starter and grower periods). A completely randomized block design was adopted and main effects (CP and Thr) were arranged in a 2 × 4 factorial approach. Optimum growth performance was achieved when broiler requirements for CP and Thr were 100% satisfied. The 110% Thr inclusion in 97.5% CP diet increased ADG, ADFI, energy intake, and protein intake (Thr, P < 0.01; quadratic, P = 0.01). The G:F (linear, P = 0.05) and energy efficiency (linear, P = 0.04) tended to decreased (Thr, P = 0.09) by increasing Thr supplementation level, whereas protein efficiency tended to increase (CP, P = 0.06) by reducing CP level. The 110% Thr inclusion in 97.5% CP diet increased eviscerated carcass weight (CP × Thr, P = 0.03) and carcass yield (Thr, P = 0.08; quadratic, P = 0.05). The reduction of CP content promoted fat abdominal deposition (CP, P = 0.05). Incremental Thr raised abdominal fat (Thr, P = 0.01; linear, P = 0.01). The 97.5% CP diets resulted in higher serum concentrations of uric acid (CP, P = 0.02), total and high- and low-density lipoprotein-linked cholesterol (CP, P≤ 0.01), and alanine aminotransferase (CP, P = 0.05) and lower (CP, P = 0.01) concentrations of triglycerides and very low density lipoproteins compared with the 100% CP diets. However, the Thr inclusion improved serum lipid profile. Irrespective of CP content, incremental Thr levels up to 120% increased (Thr, P = 0.01) broiler immune responses against Newcastle disease virus and sheep red blood cells. In order to reduce dietary CP content, strategies to increase synthetic amino acid availability, such as the use of encapsulated amino acids, should be taken into account.

Unambiguous Identification of Serine and Threonine Pyrophosphorylation Using Neutral-Loss-Triggered Electron-Transfer/Higher-Energy Collision Dissociation.

Tandem mass spectrometry (MS/MS) has emerged as the core technology for identification of post-translational modifications (PTMs). Here, we report the mass spectrometry analysis of serine and threonine pyrophosphorylation, a protein modification that has eluded detection by conventional MS/MS methods. Analysis of a set of synthesized, site-specifically modified peptides by different fragmentation techniques shows that pyrophosphorylated peptides exhibit a characteristic neutral loss pattern of 98, 178, and 196 Da, which enables the distinction between isobaric pyro- and diphosphorylated peptides. In addition, electron-transfer dissociation combined with higher energy collision dissociation (EThcD) provides exceptional data-rich MS/MS spectra for direct and unambiguous pyrophosphosite assignment. Remarkably, sufficient fragmentation of doubly charged precursors could be achieved by electron-transfer dissociation (ETD) with increased supplemental activation, without losing the labile modification. By exploiting the specific fragmentation behavior of pyrophosphorylated peptides during collision-induced dissociation (CID), a data dependent neutral-loss-triggered EThcD acquisition method was developed. This strategy enables reliable pyrophosphopeptide identification in complex samples, without compromising speed and sensitivity.

The dietary protein paradox and threonine 15 N-depletion: Pyridoxal-5'-phosphate enzyme activity as a mechanism for the δ15 N trophic level effect.

RATIONALE: Nitrogen stable isotope ratios (δ15 N values) are used to reconstruct dietary patterns, but the biochemical mechanism(s) responsible for the diet to tissue trophic level effect and its variability are not fully understood. Here δ15 N amino acid (AA) values and physiological measurements (nitrogen intake, plasma albumin concentrations, liver-reduced glutathione concentrations and leucine oxidation rates) are used to investigate increased dietary protein consumption and oxidative stress (vitamin E deficiency) in rat total plasma protein. METHODS: Using gas chromatography/combustion/isotope ratio mass spectrometry, the δ15 N values from N-pivaloyl-i-propyl esters of 15 AAs are reported for rats (n = 40) fed casein-based diets with: adequate protein (AP, 13.8%; n = 10), medium protein (MP, 25.7%; n = 10), high protein (HP, 51.3%; n = 10) or HP without vitamin E (HP-E; n = 10) for 18 weeks. RESULTS: Between the HP and AP groups, the δ15 NAA values of threonine (-4.0‰), serine (+1.4‰) and glycine (+1.2‰) display the largest differences and show significant correlations with: nitrogen intake, plasma albumin concentrations, liver-reduced glutathione concentrations and leucine oxidation rates. This indicates increased AA catabolism by the dietary induction of shared common metabolic pathways involving the enzymes threonine ammonia-lyase (EC 4.3.1.19), serine hydroxymethyltransferase (EC 2.1.2.1) and the glycine cleavage system (EC 2.1.2.10). The δ15 NAA values of the HP-E and HP groups were not found to be significantly different. CONCLUSIONS: The 15 N-depleted results of threonine are linked to increased activity of threonine ammonia-lyase, and show potential as a possible biomarker for protein intake and/or gluconeogenesis. We hypothesize that the inverse nitrogen equilibrium isotope effects of Schiff base formation, between AAs and pyridoxal-5'-phosphate cofactor enzymes, play a key role in the bioaccumulation and depletion of 15 N in the biomolecules of living organisms and contributes to the variability in the nitrogen trophic level effect. Copyright © 2017 John Wiley & Sons, Ltd.

Estimação de valores genéticos para codornas europeias em função dos níveis da relação treonina: lisina da dieta: do nascimento aos 21 dias de idade / Estimation of breeding values for European quails in function of threonine: lysine ratios of diet: from birth to 21 days of age

O presente trabalho foi realizado com o objetivo de avaliar a sensibilidade dos valores genéticos dos pesos corporais e as características de carcaças de codornas europeias às mudanças do gradiente ambiental (níveis da relação treonina com a lisina das dietas), do nascimento aos 21 dias de idade, por meio de modelos de regressão aleatória com diferentes classes de variância residual. Os dados utilizados neste estudo são provenientes de 915 codornas de corte da linhagem LF1 e 839 da linhagem LF2, pertencentes ao Programa de Melhoramento Genético da Universidade Federal dos Vales do Jequitinhonha e Mucuri. Foram avaliados os pesos corporais e os rendimentos da carcaça das aves. As sensibilidades dos valores genéticos às mudanças nos níveis da relação treonina:lisina (interação genótipo x ambiente) foram obtidas por modelos de regressão aleatória (utilizando normas de reação) por meio do programa Wombat, que utiliza o princípio da máxima verossimilhança restrita (REML). O modelo de regressão aleatória que considerou duas classes de variância residual foi o mais indicado para a maioria das análises realizadas. Verificaram-se alterações na classificação dos valores genéticos para as duas linhagens de codornas de corte estudadas. Esse comportamento indica sensibilidade de valores genéticos aditivos às mudanças nutricionais, o que caracteriza a existência de interação genótipo x ambiente. A predição dos valores genéticos deve ser feita com o mesmo nível da relação treonina:lisina da dieta com a qual as codornas serão alimentadas no sistema de produção.(AU)

This research was carried out to evaluate the sensitivity of breeding values of body weight and carcass traits in two lines of European quails (LF1 and LF2) to changes in the environment gradient (levels of threonine: lysine ratio of diets) from hatch to 21 days of age in two lines LF1 and LF2 using Random Regression Models with different classes of residual variance. Records are from 915 quails of line LF1 and 839 of line LF2 belonging to the Breeding Improvement Program of Universidade Federal dos Vales do Jequitinhonha e Mucuri. Live body weight and weights and yields of carcass, breast, and thigh and drumstick were measured. The sensitivities of breeding values to changes in threonine: lysine ratios (genotype x environment interaction) of diets were obtained by random regression models (reaction model) using the WOMBAT program using the Restricted Maximum Likelihood principle. Model considering two classes of residual variance showed the best goodness of fit. The Reaction Norms analyses indicated changes in the ranking of breeding values for both lines suggesting quails selected in one level of threonine: lysine ratio will not express all their genetic potential if fed different threonine: lysine ratio diets. This behavior indicates sensitivity of breeding values to changes in the nutrition characterizing the genotype by environment interaction. The prediction of breeding values must be performed using the same level of threonine: lysine ratio in diet the quails will be fed in the production system.(AU)

Structures and serospecificity of threonine-containing O polysaccharides of two clinical isolates belonging to the genus Proteus and their classification into O11 subserogroups.

Two clinical isolates from Polish patients, Proteus mirabilis 9B-m and Proteus genomospecies 3J-r, were found to be serologically related to P mirabilis O11. However, serological studies involving ELISA and Western blotting methods, using lipopolysaccharides (LPSs) extracted from the strains as antigens and native or adsorbed rabbit polyclonal O antisera, specific to the studied strains, revealed slight differences in the cross-reactivity and specificity of the two studied Proteus isolates, when compared to P. mirabilis O11. Two different O polysaccharides containing N-(d-galacturonoyl)-l-threonine were isolated from the LPSs of the isolates. Their structures were determined by chemical analysis and NMR spectroscopy and found to be related to the P. mirabilis O11 antigen structure established earlier, the 9B-m structure differing in the absence of the lateral glucose residue and the 3J-r structure in non-stoichiometric O-acetylation of the threonine residue only. Thus, the Proteus O11 serogroup should be divided into two subgroups: O11a, represented by the 9B-m isolate and O11a, b possessing the additional b epitope, containing the lateral residue of glucose and formed by the 3J-r isolate as well as P. mirabilis 25/57 belonging to O11 serogroup so far. O11a is the sixth new serotype found in Proteus spp. strains recently isolated from patients in central Poland.

Effects of dietary fiber on the ideal standardized ileal digestible threonine:lysine ratio for twenty-five to fifty kilogram growing gilts.

Four experiments were conducted to determine effects of fiber on the ideal Thr:Lys ratio for 25- to 50-kg gilts. In Exp. 1, the objective was to determine the requirement for standardized ileal digestible Lys for gilts from 25 to 50 kg BW. Seventy gilts (24.54 ± 3.28 kg BW) were used in a growth assay with 2 pigs per pen, 5 diets, and 7 replicate pens per diet. The 5 diets were based on corn and soybean meal and contained between 0.80 and 1.32% SID Lys. Results indicated that 1.09% SID Lys was needed to optimize ADG and G:F. In Exp. 2, the objective was to determine the standardized ileal digestibility of AA in corn, soybean meal, field peas, fish meal, and soybean hulls. Six ileal-cannulated gilts (26.5 ± 0.74 kg BW) were allotted to a 6 × 6 Latin square design with 6 diets and 6 periods. Values for standardized ileal digestibility of AA were calculated for all ingredients. In Exp. 3, the objective was to determine the effect of fiber on the ideal SID Thr:Lys ratio for gilts from 25 to 50 kg BW. A total of 192 gilts (26.29 ± 4.64 kg BW) were used in a growth assay with 2 pigs per pen and 8 replicate pens per treatment. Six low-fiber diets and 6 high-fiber diets were formulated using the same batches of ingredients as in Exp. 2. Within each level of fiber, diets with SID Thr:Lys ratios ranging from 45:100 to 90:100 were formulated using the SID values calculated in Exp. 2. In both types of diets, ADG and G:F linearly and quadratically ( < 0.05) increased as the Thr:Lys ratio increased. Regression analysis estimated the ideal SID Thr:Lys ratio at 0.66 and 0.63 for ADG and G:F, respectively, for pigs fed low-fiber diets and at 0.71 and 0.63, respectively, for pigs fed high-fiber diets. In Exp. 4, the objective was to determine the N balance in pigs fed low-fiber or high-fiber diets that were formulated to have SID Thr:Lys ratios of 45:100 or 60:100. The 4 diets were formulated using the same batches of ingredients as in Exp. 2, and the SID values determined in Exp. 2 were used in diet formulations. Thirty-six gilts (29.0 ± 0.74 kg BW) were individually housed in metabolism crates with 9 replicate pigs per diet. Retention of N (% of intake) was greater (P < 0.05) for pigs fed the low-fiber diets compared with pigs fed the high-fiber diets regardless of the Thr:Lys ratio. Results of these experiments indicate that increased fiber levels in diets fed to growing gilts increase the requirement for Thr and that diets with higher fiber levels should be formulated to a greater SID Thr:Lys ratio.