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In higher fungi, lysine is biosynthesized via the α-aminoadipate (AAA) pathway, which differs from plants, bacteria, and lower fungi. The differences offer a unique opportunity to develop a molecular regulatory strategy for the biological control of plant parasitic nematodes, based on nematode-trapping fungi. In this study, in the nematode-trapping fungus model Arthrobotrys oligospora, we characterized the core gene in the AAA pathway, encoding α-aminoadipate reductase (Aoaar), via sequence analyses and through comparing the growth, and biochemical and global metabolic profiles of the wild-type and Aoaar knockout strains. Aoaar not only has α-aminoadipic acid reductase activity, which serves fungal L-lysine biosynthesis, but it also is a core gene of the non-ribosomal peptides biosynthetic gene cluster. Compared with WT, the growth rate, conidial production, number of predation rings formed, and nematode feeding rate of the ΔAoaar strain were decreased by 40–60%, 36%, 32%, and 52%, respectively. Amino acid metabolism, the biosynthesis of peptides and analogues, phenylpropanoid and polyketide biosynthesis, and lipid metabolism and carbon metabolism were metabolically reprogrammed in the ΔAoaar strains. The disruption of Aoaar perturbed the biosynthesis of intermediates in the lysine metabolism pathway, then reprogrammed amino acid and amino acid-related secondary metabolism, and finally, it impeded the growth and nematocidal ability of A. oligospora. This study provides an important reference for uncovering the role of amino acid-related primary and secondary metabolism in nematode capture by nematode-trapping fungi, and confirms the feasibility of Aoarr as a molecular target to regulate nematode-trapping fungi to biocontrol nematodes.

Lysine crotonylation (Kcr) is a type of protein post-translational modification (PTM), which plays important roles in a variety of cellular regulation and processes. Several methods have been proposed for the identification of crotonylation. However, most of these methods can predict efficiently only on histone or non-histone protein. Therefore, this work aims to give a more balanced performance in different species, here plant (non-histone) and mammalian (histone) are involved. SVM (support vector machine) and RF (random forest) were employed in this study. According to the results of cross-validations, the RF classifier based on EGAAC attribute achieved the best predictive performance which performs competitively good as existed methods, meanwhile more robust when dealing with imbalanced datasets. Moreover, an independent test was carried out, which compared the performance of this study and existed methods based on the same features or the same classifier. The classifiers of SVM and RF could achieve best performances with 92% sensitivity, 88% specificity, 90% accuracy, and an MCC of 0.80 in the mammalian dataset, and 77% sensitivity, 83% specificity, 70% accuracy and 0.54 MCC in a relatively small dataset of mammalian and a large-scaled plant dataset respectively. Moreover, a cross-species independent testing was also carried out in this study, which has proved the species diversity in plant and mammalian.

Mutualisms, or reciprocally beneficial interspecific interactions, constitute the foundation of many ecological communities and agricultural systems. Mutualisms come in different forms, from pairwise interactions to extremely diverse communities, and they are continually challenged with exploitation by nonmutualistic community members (exploiters). Thus, understanding how mutualisms persist remains an essential question in ecology. Theory suggests that high species richness and functional redundancy could promote mutualism persistence in complex mutualistic communities. Using a yeast system (Saccharomyces cerevisiae), we experimentally show that communities with the greatest mutualist richness and functional redundancy are nearly two times more likely to survive exploitation than are simple communities. Persistence increased because diverse communities were better able to mitigate the negative effects of competition with exploiters. Thus, large mutualistic networks may be inherently buffered from exploitation.

Pyridoxine-dependent epilepsy (PDE) (OMIM 266100) is an autosomal recessive disorder of lysine metabolism secondary to antiquitin deficiency. The prototypical presentation is intractable neonatal seizures that do not respond to conventional antiseizure medication but are well controlled by pyridoxine supplementation. Atypical forms account for one-third of the PDE spectrum and may escape early diagnosis. The common atypical presentations include the prenatal onset of seizures, seizures onset as delayed as 3 years of age, autism, arrested hydrocephalus, and fetal ventriculomegaly. Herein, we describe a 9-month-old child with neonatal-onset refractory seizures who failed two short trials of pyridoxine therapy and was later diagnosed with PDE by molecular studies. Regardless of the therapeutic response, a prolonged course of pyridoxine therapy is justified to identify delayed responders in infants with drug-refractory epilepsy of no apparent etiology.

Nonribosomal depsipeptides are natural products composed of amino and hydroxy acid residues. The hydroxy acid residues often derive from α-keto acids, reduced by ketoreductase domains in the depsipeptide synthetases. Biochemistry and structures reveal the mechanism of discrimination for α-keto acids and a remarkable architecture: flanking intact adenylation and ketoreductase domains are sequences separated by >1,100 residues that form a split 'pseudoAsub' domain, structurally important for the depsipeptide module's synthetic cycle.

Microtubule-associated proteins (MAPs) are a functionally highly diverse class of proteins that help to adjust the shape and function of the microtubule cytoskeleton in space and time. For this purpose, MAPs structurally support microtubules, modulate their dynamic instability, or regulate the activity of associated molecular motors. The microtubule-binding domains of MAPs are structurally divergent, but often depend on electrostatic interactions with the negatively charged surface of the microtubule. This suggests that the surface exposure of positive charges rather than a certain structural fold is sufficient for a protein to associate with microtubules. Consistently, positively charged artificial objects have been shown to associate with microtubules and to diffuse along their lattice. Natural MAPs, however, show a more sophisticated functionality beyond lattice-diffusion. Here, we asked whether basic electrostatic interactions are sufficient to also support advanced MAP functionality. To test this hypothesis, we studied simple positively charged peptide sequences for the occurrence of typical MAP-like behavior. We found that a multivalent peptide construct featuring four lysine-alanine heptarepeats (starPEG-(KA7)4)—but not its monovalent KA7-subunits—show advanced, biologically relevant MAP-like behavior: starPEG-(KA7)4 binds microtubules in the low nanomolar range, diffuses along their lattice with the ability to switch between intersecting microtubules, and tracks depolymerizing microtubule ends. Further, starPEG-(KA7)4 promotes microtubule nucleation and growth, mediates depolymerization coupled pulling at plus ends, and bundles microtubules without significantly interfering with other proteins on the microtubule lattice (as exemplified by the motor kinesin-1). Our results show that positive charges and multivalency are sufficient to mimic advanced MAP-like behavior.

Glutaric aciduria type I (GA-I, OMIM # 231670) is an inborn error of metabolism caused by a deficiency of glutaryl-CoA dehydrogenase (GCDH). Patients develop acute encephalopathic crises (AEC) with striatal injury most often triggered by catabolic stress. The pathophysiology of GA-I, particularly in brain, is still not fully understood. We generated the first knock-in rat model for GA-I by introduction of the mutation p.R411W, the rat sequence homologue of the most common Caucasian mutation p.R402W, into the Gcdh gene of Sprague Dawley rats by CRISPR/CAS9 technology. Homozygous Gcdhki/ki rats revealed a high excretor phenotype, but did not present any signs of AEC under normal diet (ND). Exposure to a high lysine diet (HLD, 4.7%) after weaning resulted in clinical and biochemical signs of AEC. A significant increase of plasmatic ammonium concentrations was found in Gcdhki/ki rats under HLD, accompanied by a decrease of urea concentrations and a concomitant increase of arginine excretion. This might indicate an inhibition of the urea cycle. Gcdhki/ki rats exposed to HLD showed highly diminished food intake resulting in severely decreased weight gain and moderate reduction of body mass index (BMI). This constellation suggests a loss of appetite. Under HLD, pipecolic acid increased significantly in cerebral and extra-cerebral liquids and tissues of Gcdhki/ki rats, but not in WT rats. It seems that Gcdhki/ki rats under HLD activate the pipecolate pathway for lysine degradation. Gcdhki/ki rat brains revealed depletion of free carnitine, microglial activation, astroglyosis, astrocytic death by apoptosis, increased vacuole numbers, impaired OXPHOS activities and neuronal damage. Under HLD, Gcdhki/ki rats showed imbalance of intra-and extracellular creatine concentrations and indirect signs of an intracerebral ammonium accumulation. We successfully created the first rat model for GA-I. Characterization of this Gcdhki/ki strain confirmed that it is a suitable model not only for the study of pathophysiological processes, but also for the development of new ther-apeutic interventions. We further brought up interesting new insights into the pathophysiology of GA-I in brain and periphery., (c) 2021 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).

Lysine (Lys) is indispensable nutritionally, and its levels in plants are modulated by both transcriptional and post-transcriptional control during plant ontogeny. Animal glutamate receptor homologs have been detected in plants, which may participate in several plant processes through the Lys catabolic products. Interestingly, a connection between Lys and serotonin metabolism has been established recently in rice. 2-Aminoadipate, a catabolic product of Lys appears to play a critical role between serotonin accumulation and the color of rice endosperm/grain. It has also been shown that expression of some lysine-methylated proteins and genes encoding lysine-methyltransferases (KMTs) are regulated by cadmium even as it is known that Lys biosynthesis and its degradation are modulated by novel mechanisms. Three complex pathways co-exist in plants for serine (Ser) biosynthesis, and the relative preponderance of each pathway in relation to plant development or abiotic stress tolerance are being unfolded slowly. But the phosphorylated pathway of L-Ser biosynthesis (PPSB) appears to play critical roles and is essential in plant metabolism and development. Ser, which participates indirectly in purine and pyrimidine biosynthesis and plays a pivotal role in plant metabolism and signaling. Also, L-Ser has been implicated in plant responses to both biotic and abiotic stresses. A large body of information implicates Lys-rich and serine/arginine-rich (SR) proteins in a very wide array of abiotic stresses. Interestingly, a link exists between Lys-rich K-segment and stress tolerance levels. It is of interest to note that abiotic stresses largely influence the expression patterns of SR proteins and also the alternative splicing (AS) patterns. We have checked if any lncRNAs form a cohort of differentially expressed genes from the publicly available PPSB, sequence read archives of NCBI GenBank. Finally, we discuss the link between Lys and Ser synthesis, catabolism, Lys-proteins, and SR proteins during plant development and their myriad roles in response to abiotic stresses.

Summary Lysine is the main limiting essential amino acid (EAA) in the rice seeds, which is a major energy and nutrition source for humans and livestock. In higher plants, the rate‐limiting steps in lysine biosynthesis pathway are catalysed by two key enzymes, aspartate kinase (AK) and dihydrodipicolinate synthase (DHDPS), and both are extremely sensitive to feedback inhibition by lysine. In this study, two rice AK mutants (AK1 and AK2) and five DHDPS mutants (DHDPS1–DHDPS5), all single amino acid substitution, were constructed. Their protein sequences passed an allergic sequence‐based homology alignment. Mutant proteins were recombinantly expressed in Escherichia coli, and all were insensitive to the lysine analog S‐(2‐aminoethyl)‐l‐cysteine (AEC) at concentrations up to 12 mm. The AK and DHDPS mutants were transformed into rice, and free lysine was elevated in mature seeds of transgenic plants, especially those expressing AK2 or DHDPS1, 6.6‐fold and 21.7‐fold higher than the wild‐type (WT) rice, respectively. We then engineered 35A2D1L plants by simultaneously expressing modified AK2 and DHDPS1, and inhibiting rice LKR/SDH (lysine ketoglutaric acid reductase/saccharopine dehydropine dehydrogenase). Free lysine levels in two 35A2D1L transgenic lines were 58.5‐fold and 39.2‐fold higher than in WT and transgenic rice containing native AK and DHDPS, respectively. Total free amino acid and total protein content were also elevated in 35A2D1L transgenic rice. Additionally, agronomic performance analysis indicated that transgenic lines exhibited normal plant growth, development and seed appearance comparable to WT plants. Thus, AK and DHDPS mutants may be used to improve the nutritional quality of rice and other cereal grains.

Pyridoxine-dependent epilepsy (PDE) is a severe neonatal seizure disorder and is here modeled in aldh7a1 -/- zebrafish. Mutant larvae display spontaneous.., Pyridoxine-dependent epilepsy (PDE) is a rare disease characterized by mutations in the lysine degradation gene ALDH7A1 leading to recurrent neonatal seizures, which are uniquely alleviated by high doses of pyridoxine or pyridoxal 5′-phosphate (vitamin B6 vitamers). Despite treatment, neurodevelopmental disabilities are still observed in most PDE patients underlining the need for adjunct therapies. Over 60 years after the initial description of PDE, we report the first animal model for this disease: an aldh7a1-null zebrafish (Danio rerio) displaying deficient lysine metabolism and spontaneous and recurrent seizures in the larval stage (10 days postfertilization). Epileptiform electrographic activity was observed uniquely in mutants as a series of population bursts in tectal recordings. Remarkably, as is the case in human PDE, the seizures show an almost immediate sensitivity to pyridoxine and pyridoxal 5′-phosphate, with a resulting extension of the life span. Lysine supplementation aggravates the phenotype, inducing earlier seizure onset and death. By using mass spectrometry techniques, we further explored the metabolic effect of aldh7a1 knockout. Impaired lysine degradation with accumulation of PDE biomarkers, B6 deficiency, and low γ-aminobutyric acid levels were observed in the aldh7a1−/− larvae, which may play a significant role in the seizure phenotype and PDE pathogenesis. This novel model provides valuable insights into PDE pathophysiology; further research may offer new opportunities for drug discovery to control seizure activity and improve neurodevelopmental outcomes for PDE.

Biological aging research is expected to reveal modifiable molecular mechanisms that can be harnessed to slow or possibly reverse unhealthy trajectories. However, there is first an urgent need to define consensus molecular markers of healthy and unhealthy aging. Established aging hallmarks are all linked to metabolism, and a ‘rewired’ metabolic circuitry has been shown to accelerate or delay biological aging. To identify metabolic signatures distinguishing healthy from unhealthy aging trajectories, we performed nontargeted metabolomics on skeletal muscles from 2-month-old and 21-month-old mice, and after dietary and lifestyle interventions known to impact biological aging. We hypothesized that common metabolic signatures would highlight specific pathways and processes promoting healthy aging, while revealing the molecular underpinnings of unhealthy aging. Here, we report 50 metabolites that commonly distinguished aging trajectories in all cohorts, including 18 commonly reduced under unhealthy aging and 32 increased. We stratified these metabolites according to known relationships with various aging hallmarks and found the greatest associations with oxidative stress and nutrient sensing. Collectively, our data suggest interventions aimed at maintaining skeletal muscle arginine and lysine may be useful therapeutic strategies to minimize biological aging and maintain skeletal muscle health, function, and regenerative capacity in old age.

Post-translational modifications of histones by protein methyltransferases (PMTs) and histone demethylases (KDMs) play an important role in the regulation of gene expression and transcription and are implicated in cancer and many other diseases. Many of these enzymes also target various nonhistone proteins impacting numerous crucial biological pathways. Given their key biological functions and implications in human diseases, there has been a growing interest in assessing these enzymes as potential therapeutic targets. Consequently, discovering and developing inhibitors of these enzymes has become a very active and fast-growing research area over the past decade. In this review, we cover the discovery, characterization, and biological application of inhibitors of PMTs and KDMs with emphasis on key advancements in the field. We also discuss challenges, opportunities, and future directions in this emerging, exciting research field.

The main objective of this experiment was to determine the standardized ileal digestible (SID) Lys requirement of immunologically castrated (IC) and physically castrated (PC) male pigs during 3 growth phases. An additional objective was to compare the ADFI of PC and IC after the second anti-gonadotropin releasing factor (GnRF) injection. Three hundred male pigs (PIC 359 × C29), 150 each of IC and PC, were allotted to 1 of 5 treatments: 80, 90, 100, 110, or 120% of the estimated Lys requirement using the modeling program. Pigs remained on the same treatment throughout each of the 3 phases. Lysine requirements were determined at 3 stages of growth starting at a BW of 30.0 ± 0.8, 64.7 ± 1.4, and 111.9 ± 1.9 kg for IC and 32.4 ± 0.6, 69.8 ± 1.0, and 114.5 ± 1.3 kg for PC. Anti-GnRF injections were administered to IC at 11.5 and 19 wk (average BW = 96.3 ± 1.8 kg) of age. The one-slope broken line regression and quadratic plateau models were used, and the best model was selected based on the Akaike information criterion. The IC SID Lys requirements based on ADG were 1.03, 0.97, and 0.55% and for G:F, the requirements were 0.99, 0.72, and 0.55% for phases 1, 2, and 3, respectively. For PC, the SID Lys requirements based on ADG were 0.86, 0.62, and 0.47% and for G:F were 0.86, 0.58, and 0.47% for phases 1, 2, and 3, respectively. Immunologically castrated pigs had greater SID Lys requirements for phases 1 and 2 compared to PC (0.17 and 0.35% points greater for ADG and 0.13 and 0.14% points greater for G:F for phases 1 and 2, respectively). After the second injection, when IC pigs are believed to become physiologically more similar to PC pigs, the SID Lys requirements continue to be greater for IC (0.55 vs. 0.47% for both ADG and G:F). The ADFI of IC increased 4 d after the second injection relative to PC. By 2 wk post second injection, the ADFI of IC exceeded that of PC ( < 0.05). Adopting immunological castration as a management tool requires an understanding that IC need to be fed differently compared to PC, to maximize growth performance. Immunologically castrated pigs have a greater SID Lys requirement throughout the grower and finishing periods compared to the PC.

Pyridoxine (vitamin B6)-responsive epilepsies are severe forms of epilepsy that manifest as seizures immediately after birth, sometimes in utero, sometimes months, or years after birth. Seizures may be treated efficiently by life-long supplementation with pyridoxine or its biologically active form, pyridoxal phosphate, but even so patients may become intellectually disabled, for which there currently is no effective treatment. The condition may be caused by mutations in several genes (TNSALP, PIGV, PIGL, PIGO, PNPO, PROSC, ALDH7A1, MOCS2, or ALDH4A1). Mutations in ALDH7A1, MOCS2, and ALDH4A1 entail build-up of reactive aldehydes (α-aminoadipic semialdehyde, γ-glutamic semialdehyde) that may react non-enzymatically with macromolecules of brain cells. Such reactions may alter the function of macromolecules, and they may produce “advanced glycation end products” (AGEs). AGEs trigger inflammation in the brain. This understanding points to aldehyde-quenching, anti-AGE, or anti-inflammatory therapies as possible strategies to protect cognitive development and prevent intellectual disability in affected children. Studies on how aldehydes traverse cell membranes and how they affect brain function could further the development of therapies for patients with pyridoxine-responsive epilepsies.

Herein we provide a mass spectrometry-based lysine reactivity profiling strategy to monitor the ligand modulation of protein receptors, which is achieved by active dimethyl labeling of lysine residues and comparison of the alterations of labeling reactivity during ligand binding. The small-molecule ligand modulation patterns on the catechol-O-methyltransferase and the N-methyl-d-aspartate receptors have been predicted, including both binding regions and related conformational changes.

Most modern tools used to predict sites of small ubiquitin-like modifier (SUMO) binding (referred to as SUMOylation) use algorithms, chemical features of the protein, and consensus motifs. However, these tools rarely consider the influence of post-translational modification (PTM) information for other sites within the same protein on the accuracy of prediction results. This study applied the Random Forest machine learning method, as well as motif screening models and a feature selection combination mechanism, to develop a SUMOylation prediction system, referred to as SUMOgo. With regard to prediction method, PTM sites were coded as new functional features in addition to structural features, such as sequence-based binary coding, encoded chemical features of proteins, and encoded secondary structure information that is important for PTM. Twenty cycles of prediction were conducted with a 1:1 combination of positive test data and random negative data. Matthew’s correlation coefficient of SUMOgo reached 0.511, which is higher than that of current commonly used tools. This study further verified the important role of PTM in SUMOgo and includes a case study on CREB binding protein (CREBBP). The website for the final tool is http://predictor.nchu.edu.tw/SUMOgo.

Supplemental Digital Content is available in the text, Introduction: Histone post-translational modifications (PTMs) carry epigenetic information to regulate diverse cellular processes at the chromatin level. Crotonylation, one of the most important and common PTMs, plays a key role in the regulation of various biological processes. However, no study has evaluated the role of lysine crotonylation in maintenance hemodialysis patients (MHP). Methods: Here, we comparatively evaluated the crotonylation proteome of normal controls (NC) and MHP using liquid chromatography tandem mass spectrometry (LC-MS/MS) coupled with highly sensitive immune-affinity purification. Results: A total of 1109 lysine modification sites distributed on 347 proteins were identified, including 93 and 252 crotonylated upregulated and downregulated proteins, respectively. Thus, a decrease in crotonylation of histone proteins was observed in patients with kidney failure undergoing maintenance hemodialysis. Intensive bioinformatic analysis revealed that most of the crotonylated proteins were distributed in the cytoplasm, nucleus, mitochondria, and extracellular region. Gene ontology enrichment analysis showed that the crotonylated proteins were significantly enriched in the platelet alpha granule lumen, platelet degranulation, and cell adhesion molecule binding. In addition, protein domain, including fibrinogen alpha/beta/gamma chain, zinc finger, and WD40-repeat-containing domain, were significantly enriched in crotonylated proteins. Kyoto Encyclopedia of Genes and Genomes (KEGG)-based functional enrichment analysis revealed that crotonylated proteins were enriched in complement and coagulation cascades, cardiac muscle contraction, and hematopoietic cell lineage, all of which have important associations with hemodialysis complications. Conclusions: This is the first report on the global crotonylation proteome of MHP. Lysine crotonylation was found to play important regulatory roles in pathophysiological processes in MHP.

Post-translational methylation is a chemically simple modification, but regulates the function of hundreds of proteins in profound ways. This Feature Article will report on the basic aspects of protein methylation, and will offer a personal perspective on our recent efforts at making supramolecular hosts that can bind and discriminate among post-translationally methylated partners. The article highlights several general lessons drawn from these efforts and related work by other groups. It also describes some ways in which supramolecular approaches are inherently well suited to provide tools that drive new research in the life sciences.

Two experiments were conducted to estimate the standardized ileal digestible (SID) Trp:Lys ratio requirement for growth performance of nursery pigs. Experimental diets were formulated to ensure that lysine was the second limiting AA throughout the experiments. In Exp. 1 (6 to 10 kg BW), 255 nursery pigs (PIC 327 × 1050, initially 6.3 ± 0.15 kg, mean ± SD) arranged in pens of 6 or 7 pigs were blocked by pen weight and assigned to experimental diets (7 pens/diet) consisting of SID Trp:Lys ratios of 14.7%, 16.5%, 18.4%, 20.3%, 22.1%, and 24.0% for 14 d with 1.30% SID Lys. In Exp. 2 (11 to 20 kg BW), 1,088 pigs (PIC 337 × 1050, initially 11.2 kg ± 1.35 BW, mean ± SD) arranged in pens of 24 to 27 pigs were blocked by average pig weight and assigned to experimental diets (6 pens/diet) consisting of SID Trp:Lys ratios of 14.5%, 16.5%, 18.0%, 19.5%, 21.0%, 22.5%, and 24.5% for 21 d with 30% dried distillers grains with solubles and 0.97% SID Lys. Each experiment was analyzed using general linear mixed models with heterogeneous residual variances. Competing heteroskedastic models included broken-line linear (BLL), broken-line quadratic (BLQ), and quadratic polynomial (QP). For each response, the best-fitting model was selected using Bayesian information criterion. In Exp. 1 (6 to 10 kg BW), increasing SID Trp:Lys ratio linearly increased ( 0.05) ADG and G:F. For ADG, the best-fitting model was a QP in which the maximum ADG was estimated at 23.9% (95% confidence interval [CI]: [ 24.0%]) SID Trp:Lys ratio. For G:F, the best-fitting model was a BLL in which the maximum G:F was estimated at 20.4% (95% CI: [14.3%, 26.5%]) SID Trp:Lys. In Exp. 2 (11 to 20 kg BW), increasing SID Trp:Lys ratio increased ( 0.05) ADG and G:F in a quadratic manner. For ADG, the best-fitting model was a QP in which the maximum ADG was estimated at 21.2% (95% CI: [20.5%, 21.9%]) SID Trp:Lys. For G:F, BLL and BLQ models had comparable fit and estimated SID Trp:Lys requirements at 16.6% (95% CI: [16.0%, 17.3%]) and 17.1% (95% CI: [16.6%, 17.7%]), respectively. In conclusion, the estimated SID Trp:Lys requirement in Exp. 1 ranged from 20.4% for maximum G:F to 23.9% for maximum ADG, whereas in Exp. 2 it ranged from 16.6% for maximum G:F to 21.2% for maximum ADG. These results suggest that standard recommendations may underestimate the SID Trp:Lys requirement for nursery pigs from 11 to 20 kg BW.

Two experiments were conducted to estimate the standardized ileal digestible valine:lysine (SID Val:Lys) dose response effects in 25- to 45-kg pigs under commercial conditions. In experiment 1, a total of 1,134 gilts (PIC 337 × 1050), initially 31.2 kg ± 2.0 kg body weight (BW; mean ± SD) were used in a 19-d growth trial with 27 pigs per pen and seven pens per treatment. In experiment 2, a total of 2,100 gilts (PIC 327 × 1050), initially 25.4 ± 1.9 kg BW were used in a 22-d growth trial with 25 pigs per pen and 12 pens per treatment. Treatments were blocked by initial BW in a randomized complete block design. In experiment 1, there were a total of six dietary treatments with SID Val at 59.0, 62.5, 65.9, 69.6, 73.0, and 75.5% of Lys and for experiment 2 there were a total of seven dietary treatments with SID Val at 57.0, 60.6, 63.9, 67.5, 71.1, 74.4, and 78.0% of Lys. Experimental diets were formulated to ensure that Lys was the second limiting amino acid throughout the experiments. Initially, linear mixed models were fitted to data from each experiment. Then, data from the two experiments were combined to estimate dose-responses using a broken-line linear ascending (BLL) model, broken-line quadratic ascending (BLQ) model, or quadratic polynomial (QP). Model fit was compared using Bayesian information criterion (BIC). In experiment 1, ADG increased linearly (P = 0.009) with increasing SID Val:Lys with no apparent significant impact on G:F. In experiment 2, ADG and ADFI increased in a quadratic manner (P < 0.002) with increasing SID Val:Lys whereas G:F increased linearly (P < 0.001). Overall, the best-fitting model for ADG was a QP, whereby the maximum mean ADG was estimated at a 73.0% (95% CI: [69.5, >78.0%]) SID Val:Lys. For G:F, the overall best-fitting model was a QP with maximum estimated mean G:F at 69.0% (95% CI: [64.0, >78.0]) SID Val:Lys ratio. However, 99% of the maximum mean performance for ADG and G:F were achieved at, 68% and 63% SID Val:Lys ratio, respectively. Therefore, the SID Val:Lys requirement ranged from73.0% for maximum ADG to 63.2% SID Val:Lys to achieve 99% of maximum G:F in 25- to 45-kg BW pigs.

Despite better knowledge of its genetic basis, pancreatic cancer is still highly lethal with very few therapeutic options. In this review, we discuss the potential impact of epigenetic therapies, focusing on lysine methylation signaling and its implication in pancreatic cancer.Protein lysine methylation, a key mechanism of posttranslational modifications of histone proteins, has emerged as a major cell signaling mechanism regulating physiologic and pathologic processes including cancer. This finely tuned and dynamic signaling mechanism is regulated by lysine methyltransferases (KMT), lysine demethylases (KDM) and signal transducers harboring methyl-binding domains. Recent evidence demonstrates that overexpression of cytoplasmic KMT and resulting enhanced lysine methylation is a reversible event that enhances oncogenic signaling through the Ras and Mitogen-Activated Protein Kinases pathway in pancreatic cancer, opening perspectives for new anticancer chemotherapeutics aimed at controlling these activities.The development of potent and specific inhibitors of lysine methylation signaling may represent a hitherto largely unexplored avenue for new forms of targeted therapy in cancer, with great potential for yet hard-to-treat cancers such as pancreatic cancer.

Increasing evidence suggests that spermatogonial stem cells (SSCs) have great clinical potential to give rise to a variety of cell types besides all spermatogenic lineage cells. The development of an efficient method for long-term culture of highly-pure SSCs is essential for further studies related to SSC biological events. Here, we describe an in vitro culture system obtaining mouse SSC cultures of high purity, viability, and proliferation. For establishing long-term cultures of SSCs, we mainly focused on isolation procedures and culture conditions. These included co-coating of extracellular substrates, that is, poly-L-lysine (PLL) and laminin, as well as combinatiorial use of three milder enzymes and simultaneously less trypsin to minimize enzyme-mediated degradation of SSCs. Furthermore, a unique purification procedure was performed to effectively eliminate contaminating non-SSCs. Finally, a critical step is to ensure SSC maintenance and expansion by utilizing optimal culture medium. Obtained data suggest that applying our optimally modified method, SSCs can be cultured for over 90 days with high purity (around 93.5%). Moreover, SSCs isolated and expanded using our protocol fulfills all criteria of SSCs without losing their stemness-characterized by SSC-phenotypic gene expression and long-term self-renewal. This study describes for the first time a protocol allowing isolation and expansion of SSCs suitable for numerous studies related to SSC-based clinical therapies of various diseases. J. Cell. Physiol. 230: 1365–1375, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company

Liquid–liquid phase separation (LLPS) of proteins containing intrinsically disordered regions (IDRs) has been proposed as a mechanism underlying the formation of membrane-less organelles. Tight regulation of IDR behavior is essential to ensure that LLPS only takes place when necessary. Here, we report that IDR acetylation/deacetylation regulates LLPS and assembly of stress granules (SGs), membrane-less organelles forming in response to stress. Acetylome analysis revealed that the RNA helicase DDX3X, an important component of SGs, is a novel substrate of the deacetylase HDAC6. The N-terminal IDR of DDX3X (IDR1) can undergo LLPS in vitro, and its acetylation at multiple lysine residues impairs the formation of liquid droplets. We also demonstrated that enhanced LLPS propensity through deacetylation of DDX3X-IDR1 by HDAC6 is necessary for SG maturation, but not initiation. Our analysis provides a mechanistic framework to understand how acetylation and deacetylation of IDRs regulate LLPS spatiotemporally, and impact membrane-less organelle formation in vivo. HDAC6 modulates acetylation at multiple lysine residues in the N-terminal intrinsically disordered region of RNA helicase DDX3X to regulate liquid–liquid phase separation and stress granule maturation.

Penicillin-binding proteins (PBPs) are enzymes involved in the assembly of the bacterial cell wall, a major target for antibiotics. These proteins are classified by mass into high-molecular-weight PBPs, which are transpeptidases that form peptidoglycan cross-links, and low-molecular-weight PBPs, which are typically hydrolases. We report a functionally unique family of low-molecular-weight PBPs that act as transpeptidases rather than hydrolases, but they do not cross-link peptidoglycan. We show that these PBPs can exchange d-amino acids bearing chemical tags or affinity handles into peptidoglycan precursors, including Lipid II, enabling biochemical studies of proteins involved in cell wall assembly. We report that, in two organisms, the PBPs incorporate lysine into cellular peptidoglycan and that, further, the PBPs have the unprecedented ability to transfer the primary e-amine of lysine to peptidoglycan.

Ubiquitin and ubiquitin-like modifications are central to virtually all cellular signaling pathways. They occur primarily on lysine residues of target proteins and stimulate a large number of downstream signals. The diversity of these signals depends on the type, location and dynamics of the modification, but the role of the exact site of modification and the selectivity for specific lysines are poorly understood. Here we review the current literature on lysine specificity in these modifications, and we highlight the known signaling mechanisms and the open questions that pose future challenges to ubiquitin research.

The aim of the present study was to determine the minimum requirement of Ile in young pigs, enabling feeding of balanced low-CP diets. Most previous studies have used experimental diets that included blood cells, which are particularly high in Leu and known to antagonize the use of Ile. One week after weaning at d 28, 100 crossbred female pigs weighing 7.9 ± 0.7 kg were allocated to 1 of 5 dietary treatments. Diets were formulated to contain 1.15 g standardized ileal digestible (SID) Lys/MJ NE and were free of blood cells. The SID Ile was 0.42, 0.47,0.53, 0.58, and 0.62 relative to Lys. The other indispensable AA were supplied according to requirements. Representative samples from the 5 diets were analyzed in 4 replicates at 3 different laboratories. The pigs were fed ad libitum and individually housed in 7 identical rooms during a 21-d period. At d 0, 7, 14, and 21, the pigs were weighed, and feed intake was determined. At d 15, blood samples were collected from the jugular vein to determine the plasma urea and free AA content. There were differences among the 3 laboratories in theanalyzed content of several AA, and also Ile and Lys showed a large variation within the diets, which may cause variation in published requirement estimates.The concentration of Ile in plasma increased linearly (P < 0.01), and Lys in plasma decreased linearly (P = 0.02) with increasing SID Ile:Lys. A tendency for a linear decrease in plasma concentration was found for Thr (P = 0.10). Both ADFI and ADG were reduced when Ile was supplied above the Ile requirement estimate.Quadratic regression curves on ADFI, ADG, and G:F all showed the maximum at 0.52 SID Ile:Lys. Modeling with 2-sloped quadratic broken-line curves showed themaximum at 0.50, 0.53, and 0.54 SID Ile:Lys for ADFI, ADG, and G:F, respectively. In conclusion, the average estimation of requirement in this dose-response study using blood cell–free diets was 0.52 SID Ile:Lys during a 21-d experimental period from 8 kg BW.

Formation of cortical connections requires the precise coordination of numerous discrete phases. This is particularly significant with regard to the corpus callosum, whose development undergoes several dynamic stages including the crossing of axon projections, elimination of exuberant projections, and myelination of established tracts. To comprehensively characterize the molecular events in this dynamic process, we set to determine the distinct temporal expression of proteins regulating the formation of the corpus callosum and their respective developmental functions. Mass spectrometry-based proteomic profiling was performed on early postnatal mouse corpus callosi, for which limited evidence has been obtained previously, using stable isotope of labeled amino acids in mammals (SILAM). The analyzed corpus callosi had distinct proteomic profiles depending on age, indicating rapid progression of specific molecular events during this period. The proteomic profiles were then segregated into five separate clusters, each with distinct trajectories relevant to their intended developmental functions. Our analysis both confirms many previously-identified proteins in aspects of corpus callosum development, and identifies new candidates in understudied areas of development including callosal axon refinement. We present a valuable resource for identifying new proteins integral to corpus callosum development that will provide new insights into the development and diseases afflicting this structure.

Three experiments were conducted separately to estimate the digestible Lys (dig. Lys) requirements of Cobb × Cobb 500 male broilers using different statistical models. For each experiment, 1,200 chicks were housed in 48 floor pens in a completely randomized design with 6 treatments and 8 replicates. Broilers were fed diets with increasing dig. Lys levels from 1 to 12 d (Exp. 1), from 12 to 28 d (Exp. 2), and 28 to 42 d (Exp. 3). Increasing dig. Lys levels were equally spaced from 0.97 to 1.37% in Exp. 1, 0.77 to 1.17% in Exp. 2, and 0.68 to 1.07% in Exp. 3. The lowest dig. Lys diets were not supplemented with L-Lysine and all other essential AA met or exceeded recommendations. In Exp. 3, six birds per pen were randomly selected from each replication to evaluate carcass and breast yields. Digestible Lys requirements were estimated by quadratic polynomial (QP), linear broken-line (LBL), quadratic broken-line (QBL), and exponential asymptotic (EA) models. Overall, dig. Lys requirements varied among response variables and statistical models. Increasing dietary dig. Lys had a positive effect on BW, carcass and breast yields. Levels of dig. Lys that optimized performance using QP, LBL, QBL, and EA models were 1.207, 1.036, 1.113, and 1.204% for BWG and 1.190, 1.027, 1.100, and 1.172% for FCR in Exp. 1; 1.019, 0.853, 0.944; 1.025% for BWG and 1.050, 0.879, 1.032, and 1.167% for FCR in Exp. 2; and 0.960, 0.835, 0.933, and 1.077% for BWG, 0.981, 0.857, 0.963, and 1.146% for FCR in Exp. 3. The QP, LBL, QBL, and EA also estimated dig. Lys requirements as 0.941, 0.846, 0.925, and 1.070% for breast meat yield in Exp. 3. In conclusion, Lys requirements vary greatly according to the statistical analysis utilized; therefore, the origin of requirement estimation must be taken into account in order to allow adequate comparisons between references.

Ubiquitination is a post-translational modification that creates versatility in cell signalling, in part because eight biochemically different inter-ubiquitin linkages can be formed through the seven internal lysine residues of ubiquitin or its amino-terminal methionine. The latter, referred to as linear or M1 linkage, is created by the linear ubiquitin chain assembly complex (LUBAC). Previously, K63 linkages were thought to be exclusively responsible for ubiquitin-mediated nondegradative functions. It now emerges, however, that M1 ubiquitination is crucial in various pathways, and that generation of a physiological signalling output requires cooperation between different ubiquitin linkage types. Here, we review the currently known functions of LUBAC and M1 ubiquitination, discuss promising future research directions into their functions, and how this may reveal novel therapeutic opportunities for diseases with perturbed linear ubiquitination.

Numerous studies in multiple systems support that histone H3 lysine 36 di-methylation (H3K36me2) is associated with transcriptional activation, however the underlying mechanisms are not well defined. Here we show that the H3K36me2 chromatin mark written by the ASH1L histone methyltransferase is preferentially bound in vivo by LEDGF, an MLL-associated protein that co-localizes with MLL, ASH1L and H3K36me2 on chromatin genome wide. Furthermore, ASH1L facilitates recruitment of LEDGF and wild type MLL proteins to chromatin at key leukemia target genes, and is a crucial regulator of MLL-dependent transcription and leukemic transformation. Conversely KDM2A, an H3K36me2 demethylase and Polycomb-group silencing protein, antagonizes MLL-associated leukemogenesis. Our studies are the first to provide a basic mechanistic insight into epigenetic interactions wherein placement, interpretation and removal of H3K36me2 contribute to the regulation of gene expression and MLL leukemia, and suggest ASH1L as a novel target for therapeutic intervention.

Lysine degradation through the saccharopine pathway has been shown only in plants and animals. Here, we show that bacteria possess the genes encoding lysine-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH). In Silicibacter, the contiguous lkr and sdh genes are interspersed, in another frame, by a polypeptide of unknown function. The bacterial enzyme does not contain the 110-amino-acid interdomain (ID) that intersperses the LKR and SDH domains of the plant enzyme. The ID was found in Cyanobacteria interspersing polypeptides without similarities and activities of LKR and SDH. The LKR/SDH bifunctional polypeptide of animals and plants may have arisen from a α-proteobacterium with a configuration similar to that of Silicibacter, whereas the ID in the plant enzyme may have been inherited from Cyanobacteria.

Four experiments were conducted to examine the effect of porcine circovirus type 2 (PCV2) vaccination on the response of growing and finishing pigs (PIC 337 × 1050) to increasing dietary Lys. Experiments 1 and 2 evaluated 38- to 65-kg gilts and barrows, respectively, and Exp. 3 and 4 evaluated 100- to 120-kg gilts and barrows, respectively. Gilts and barrows were housed separately in different barns. Treatments were allotted in a completely randomized design into 2 × 4 factorials with 2 PCV2 treatments (PCV2-vaccinated and nonvaccinated) and 4 standardized ileal digestible (SID) Lys:ME ratios (2.24, 2.61, 2.99, and 3.36 g/Mcal in Exp. 1 and 2 and 1.49, 1.86, 2.23, and 2.61 g/Mcal in Exp. 3 and 4) within each experiment. There were 5 pens per treatment. At the start of Exp. 1 and 2, there were more pigs per pen (P 0.10) were observed in any of the 4 studies. In Exp. 1 and 2, PCV2-vaccinated pigs had increased (P < 0.001) ADG compared with nonvaccinated pigs. The growth response was primarily due to increases in ADFI, which suggests that vaccinated pigs have a greater Lys requirement (g/d) than nonvaccinated pigs. In Exp. 1, increasing the SID Lys:ME ratio increased (quadratic; P < 0.04) ADG and G:F, with pigs fed the 2.99 g/Mcal ratio having the greatest ADG and G:F. In Exp. 2, increasing the SID Lys:ME ratio improved (linear; P < 0.001) G:F. In Exp. 3, ADG and G:F increased (P < 0.05) in a quadratic manner as the SID Lys:ME ratio fed increased. In Exp. 4, increasing the SID Lys:ME ratio increased ADG (linear; P < 0.001) and G:F (quadratic; P = 0.03). Although PCV2 vaccination improved growth, the corresponding increase in ADFI did not increase the optimal SID Lys:ME ratio for growing and finishing barrows and gilts.

In this issue of Molecular Cell, Huang et al. (2015) describe a nuclear pool of LC3 that is deacetylated during starvation, leading to redistribution of this essential autophagy regulator to the cytoplasm, where it can initiate autophagy.

Heat treatment of soybean meal (SBM) is necessary to reduce the concentration of trypsin inhibitors, but excessive heat treatment may reduce AA concentration and digestibility because AA can be destroyed by the Maillard reaction. The objective of this experiment was to determine the effects of heat treatment of SBM on apparent ileal digestibility and standardized ileal digestibility (SID) of AA by growing pigs. A source of conventional dehulled SBM (48.5% CP) was divided into 4 batches. One batch was not additionally heated, 1 batch was autoclaved at 125°C for 15 min, 1 batch was autoclaved at 125°C for 30 min, and 1 batch was oven-dried at 125°C for 30 min. Four SBM-cornstarch diets were formulated, and each of the 4 batches of SBM was used as the sole source of dietary AA in 1 diet. A N-free diet was used to estimate basal endogenous losses of AA. Ten growing barrows with an initial BW of 25.3 ± 2.0 kg were individually fitted with a T-cannula in the distal ileum. Pigs were allotted to treatments in a replicated 5 × 5 balanced Latin square design with 5 diets and 5 periods. Each period lasted 7 d, and ileal digesta were collected on d 6 and 7 of each period. Results of the experiment indicated that the apparent ileal digestibility and SID of CP and all AA decreased linearly (P 0.10) the SID of CP and AA in the SBM or the furosine concentration, and the color in the oven-dried sample indicated that this sample was not heat damaged. In conclusion, the digestibility of all AA in autoclaved SBM is linearly reduced as the autoclaving time increases from 0 to 30 min. The reason for these changes is most likely that autoclaving at 125°C results in Maillard reactions in SBM.

The enzymes responsible for the conversion of N(alpha)-[(benzyloxy)carbonyl]-D-lysine (N(alpha)-Z-D-lysine) to N(alpha)-Z-D-aminoadipic acid (N(alpha)-Z-D-AAA) by Rhodococcus sp. AIU Z-35-1 were identified. N(alpha)-Z-D-Lysine was first converted to N(alpha)-Z-D-aminoadipic delta-semialdehyde (N(alpha)-Z-D-AASA) by D-specific amino acid deaminase, whereas N(alpha)-Z-L-lysine was converted to N(alpha)-Z-L-AASA by L-specific amino acid oxidase. The resulting N(alpha)-Z-D-AASA was then converted to N(alpha)-Z-D-AAA by the same aldehyde dehydrogenase that is responsible for N(alpha)-Z-L-AASA oxidation. The product amount of the D-specific amino acid deaminase reached the maximum at one day of cultivation in the L-lysine medium. The aldehyde dehydrogenase reached the maximum at three days of cultivation.

Four experiments were conducted to determine the effects of standardized ileal digestible (SID) Lys:calorie (Lys:Mcal) ratio on growth perfor- mance of 10- to 20-kg pigs of 2 different genotypes. Experiment 1 (360 pigs, average BW = 10.2 kg; source

The insect mushroom bodies (MBs) are multimodal signal processing centers and are essential for olfactory learning. Electrophysiological recordings from the MBs' principal component neurons, the Kenyon cells (KCs), showed a sparse representation of olfactory signals. It has been proposed that the intrinsic and synaptic properties of the KC circuitry combine to reduce the firing of action potentials and to generate relatively brief windows for synaptic integration in the KCs, thus causing them to operate as coincidence detectors. To better understand the ionic mechanisms that mediate the KC intrinsic firing properties, we used whole cell patch-clamp recordings from KCs in the adult, intact brain of Periplaneta americana to analyze voltage- and/or Ca2+-dependent inward ( ICa, INa) and outward currents [ IA, IK(V), IK,ST, IO(Ca)]. In general the currents had properties similar to those of currents in other insect neurons. Certain functional parameters of ICaand IO(Ca), however, had unusually high values, allowing them to assist sparse coding. ICahad a low-activation threshold and a very high current density compared with those of ICain other insect neurons. Together these parameters make ICasuitable for boosting and sharpening the excitatory postsynaptic potentials as reported in previous studies. IO(Ca)also had a large current density and a very depolarized activation threshold. In combination, the large ICaand IO(Ca)are likely to mediate the strong spike frequency adaptation. These intrinsic properties of the KCs are likely to be supported by their tonic, inhibitory synaptic input, which was revealed by specific GABA antagonists and which contributes significantly to the hyperpolarized membrane potential at rest.

beta-Catenin plays an important role in development and tumorigenesis. However, the effect of a key acetyltransferase p300/CBP-associated factor (PCAF) on beta-catenin signaling is largely unknown. In this study, we found PCAF could increase the beta-catenin transcriptional activity, induce its nuclear translocation, and up-regulate its protein level by inhibiting its ubiquitination and improving its stability. Further studies showed that PCAF directly binds to and acetylates beta-catenin. The key ubiquitination sites Lys-19 and Lys-49 of beta-catenin were shown as the critical residues for PCAF-induced acetylation and stabilization. Knockdown of PCAF in colon cancer cells markedly reduced the protein level, transcriptional activity, and acetylation level of beta-catenin; promoted cell differentiation; inhibited cell migration; and repressed xenografted tumorigenesis and tumor growth in nude mice. All these data demonstrate that PCAF acetylates beta-catenin and regulates its stability, and they raise the prospect that therapies targeting PCAF may be of clinical use in beta-catenin-driven diseases, such as colon cancer.

GABA depolarizes immature cortical neurons. However, whether GABA excites immature neocortical neurons and drives network oscillations as in other brain structures remains controversial. Excitatory actions of GABA depend on three fundamental parameters: the resting membrane potential ( Em), reversal potential of GABA ( EGABA), and threshold of action potential generation ( Vthr). We have shown recently that conventional invasive recording techniques provide an erroneous estimation of these parameters in immature neurons. In this study, we used noninvasive single N-methyl-d-aspartate and GABA channel recordings in rodent brain slices to measure both Em and EGABA in the same neuron. We show that GABA strongly depolarizes pyramidal neurons and interneurons in both deep and superficial layers of the immature neocortex (P2–P10). However, GABA generates action potentials in layer 5/6 (L5/6) but not L2/3 pyramidal cells, since L5/6 pyramidal cells have more depolarized resting potentials and more hyperpolarized Vthr. The excitatory GABA transiently drives oscillations generated by L5/6 pyramidal cells and interneurons during development (P5–P12). The NKCC1 co-transporter antagonist bumetanide strongly reduces [Cl−]i, GABA-induced depolarization, and network oscillations, confirming the importance of GABA signaling. Thus a strong GABA excitatory drive coupled with high intrinsic excitability of L5/6 pyramidal neurons and interneurons provide a powerful mechanism of synapse-driven oscillatory activity in the rodent neocortex in vitro. In the companion paper, we show that the excitatory GABA drives layer-specific seizures in the immature neocortex.

Objective: To report the importance of a rare organic acid metabolic disorder, L-2-hydroxyglutaric aciduria, and its characteristic neuroimaging cerebral white matter abnormalities in a case of epilepsy. Subject and Methods: A 19-year-old male presented with an 11-year history consisting of school failures, intellectual deterioration and generalized tonic-clonic convulsions. Results: Neurological examination showed mental subnormality, mild dysarthria and bilateral pyramidal signs. Computed tomography and magnetic resonance imaging (MRI) of the brain showed characteristic white matter lesions, suggestive of L-2-hydroxyglutaric aciduria. The diagnosis of this disease was confirmed by elevated urinary concentrations of L-2-hydroxyglutaric acid. The epilepsy was partially controlled with antiepileptic drugs. Conclusion: This report indicates the importance of routine examination of urinary organic acids in children and young adults presenting with chronic encephalopathy and epilepsy with characteristic MRI white matter lesions. L-2-hydroxyglutaric aciduria should be considered as one of the differential diagnoses of epilepsy.

Differences in lysine and arginine requirements among various species such as omnivores (humans, pigs, rats, dogs), carnivores (cats), herbivores (rabbits, horses), ruminants (cattle), poultry, and fish, are covered in detail in this article. Although lysine is classified as an indispensable amino acid across species, the classification of arginine as either an indispensable or dispensable amino acid is more ambiguous because of differences among species in rates of de novo arginine synthesis. Because lysine is most often the limiting amino acid in the diet, its requirement has been extensively studied. By use of the ideal protein concept, the requirements of the other indispensable amino acids can be extrapolated from the lysine requirement. The successful use of this concept in pigs is compared with potential application of the ideal protein concept in humans. The current dietary arginine requirement varies widely among species, with ruminants, rabbits, and rats having relatively low requirements and carnivores, fish, and poultry having high requirements. Interspecies differences in metabolic arginine utilization and reasons for different rates of de novo arginine synthesis are reviewed in detail, as these are the primary determinants of the dietary arginine requirement. There is presently no dietary requirement for humans of any age, although this needs to be reassessed, particularly in neonates. A thorough understanding of the factors contributing to the lysine and arginine requirements in different species will be useful in our understanding of human amino acid requirements.