A plant effector-triggered immunity signaling sector is inhibited by pattern-triggered immunity.

Since signaling machineries for two modes of plant-induced immunity, pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), extensively overlap, PTI and ETI signaling likely interact. In an Arabidopsis quadruple mutant, in which four major sectors of the signaling network, jasmonate, ethylene, PAD4, and salicylate, are disabled, the hypersensitive response (HR) typical of ETI is abolished when the Pseudomonas syringae effector AvrRpt2 is bacterially delivered but is intact when AvrRpt2 is directly expressed in planta These observations led us to discovery of a network-buffered signaling mechanism that mediates HR signaling and is strongly inhibited by PTI signaling. We named this mechanism the ETI-Mediating and PTI-Inhibited Sector (EMPIS). The signaling kinetics of EMPIS explain apparently different plant genetic requirements for ETI triggered by different effectors without postulating different signaling machineries. The properties of EMPIS suggest that information about efficacy of the early immune response is fed back to the immune signaling network, modulating its activity and limiting the fitness cost of unnecessary immune responses.

MgO-Template Synthesis of Extremely High Capacity Hard Carbon for Na-Ion Battery.

Extremely high capacity hard carbon for Na-ion battery, delivering 478 mAh g-1 , is successfully synthesized by heating a freeze-dried mixture of magnesium gluconate and glucose by a MgO-template technique. Influences of synthetic conditions and nano-structures on electrochemical Na storage properties in the hard carbon are systematically studied to maximize the reversible capacity. Nano-sized MgO particles are formed in a carbon matrix prepared by pre-treatment of the mixture at 600 °C. Through acid leaching of MgO and carbonization at 1500 °C, resultant hard carbon demonstrates an extraordinarily large reversible capacity of 478 mAh g-1 with a high Coulombic efficiency of 88 % at the first cycle.

Exogenous Treatment with Glutamate Induces Immune Responses in Arabidopsis.

Plant resistance inducers (PRIs) are compounds that protect plants from diseases by activating immunity responses. Exogenous treatment with glutamate (Glu), an important amino acid for all living organisms, induces resistance against fungal pathogens in rice and tomato. To understand the molecular mechanisms of Glu-induced immunity, we used the Arabidopsis model system. We found that exogenous treatment with Glu induces resistance against pathogens in Arabidopsis. Consistent with this, transcriptome analyses of Arabidopsis seedlings showed that Glu significantly induces the expression of wound-, defense-, and stress-related genes. Interestingly, Glu activates the expression of genes induced by pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns at much later time points than the flg22 peptide, which is a bacterial-derived PAMP. The Glu receptor-like (GLR) proteins GLR3.3 and GLR3.6 are involved in the early expression of Glu-inducible genes; however, the sustained expression of these genes does not require the GLR proteins. Glu-inducible gene expression is also not affected by mutations in genes that encode PAMP receptors (EFR, FLS2, and CERK1), regulators of pattern-triggered immunity (BAK1, BKK1, BIK1, and PBL1), or a salicylic acid biosynthesis enzyme (SID2). The treatment of roots with Glu activates the expression of PAMP-, salicylic acid-, and jasmonic acid-inducible genes in leaves. Moreover, the treatment of roots with Glu primes chitin-induced responses in leaves, possibly through transcriptional activation of LYSIN-MOTIF RECEPTOR-LIKE KINASE 5 (LYK5), which encodes a chitin receptor. Because Glu treatment does not cause discernible growth retardation, Glu can be used as an effective PRI.

Exogenous proteinogenic amino acids induce systemic resistance in rice.

BACKGROUND: Plant immune responses can be induced by endogenous and exogenous signaling molecules. Recently, amino acids and their metabolites have been reported to affect the plant immune system. However, how amino acids act in plant defense responses has yet to be clarified. Here, we report that treatment of rice roots with amino acids such as glutamate (Glu) induced systemic disease resistance against rice blast in leaves. RESULTS: Treatment of roots with Glu activated the transcription of a large variety of defense-related genes both in roots and leaves. In leaves, salicylic acid (SA)-responsive genes, rather than jasmonic acid (JA) or ethylene (ET)-responsive genes, were induced by this treatment. The Glu-induced blast resistance was partially impaired in rice plants deficient in SA signaling such as NahG plants expressing an SA hydroxylase, WRKY45-knockdown, and OsNPR1-knockdown plants. The JA-deficient mutant cpm2 exhibited full Glu-induced blast resistance. CONCLUSIONS: Our results indicate that the amino acid-induced blast resistance partly depends on the SA pathway but an unknown SA-independent signaling pathway is also involved.

Dual regulation of gene expression mediated by extended MAPK activation and salicylic acid contributes to robust innate immunity in Arabidopsis thaliana.

Network robustness is a crucial property of the plant immune signaling network because pathogens are under a strong selection pressure to perturb plant network components to dampen plant immune responses. Nevertheless, modulation of network robustness is an area of network biology that has rarely been explored. While two modes of plant immunity, Effector-Triggered Immunity (ETI) and Pattern-Triggered Immunity (PTI), extensively share signaling machinery, the network output is much more robust against perturbations during ETI than PTI, suggesting modulation of network robustness. Here, we report a molecular mechanism underlying the modulation of the network robustness in Arabidopsis thaliana. The salicylic acid (SA) signaling sector regulates a major portion of the plant immune response and is important in immunity against biotrophic and hemibiotrophic pathogens. In Arabidopsis, SA signaling was required for the proper regulation of the vast majority of SA-responsive genes during PTI. However, during ETI, regulation of most SA-responsive genes, including the canonical SA marker gene PR1, could be controlled by SA-independent mechanisms as well as by SA. The activation of the two immune-related MAPKs, MPK3 and MPK6, persisted for several hours during ETI but less than one hour during PTI. Sustained MAPK activation was sufficient to confer SA-independent regulation of most SA-responsive genes. Furthermore, the MPK3 and SA signaling sectors were compensatory to each other for inhibition of bacterial growth as well as for PR1 expression during ETI. These results indicate that the duration of the MAPK activation is a critical determinant for modulation of robustness of the immune signaling network. Our findings with the plant immune signaling network imply that the robustness level of a biological network can be modulated by the activities of network components.

Decomposition of dynamic transcriptomic responses during effector-triggered immunity reveals conserved responses in two distinct plant cell populations.

Rapid plant immune responses in the appropriate cells are needed for effective defense against pathogens. Although transcriptome analysis is often used to describe overall immune responses, collection of transcriptome data with sufficient resolution in both space and time is challenging. We reanalyzed public Arabidopsis time-course transcriptome data obtained after low-dose inoculation with a Pseudomonas syringae strain expressing the effector AvrRpt2, which induces effector-triggered immunity in Arabidopsis. Double-peak time-course patterns are prevalent among thousands of upregulated genes. We implemented a multi-compartment modeling approach to decompose the double-peak pattern into two single-peak patterns for each gene. The decomposed peaks reveal an "echoing" pattern: the peak times of the first and second peaks correlate well across most upregulated genes. We demonstrated that the two peaks likely represent responses of two distinct cell populations that respond either cell autonomously or indirectly to AvrRpt2. Thus, the peak decomposition has extracted spatial information from the time-course data. The echoing pattern also indicates a conserved transcriptome response with different initiation times between the two cell populations despite different elicitor types. A gene set highly overlapping with the conserved gene set is also upregulated with similar kinetics during pattern-triggered immunity. Activation of a WRKY network via different entry-point WRKYs can explain the similar but not identical transcriptome responses elicited by different elicitor types. We discuss potential benefits of the properties of the WRKY activation network as an immune signaling network in light of pressure from rapidly evolving pathogens.

New risk stratification for adjuvant nivolumab for high-risk muscle-invasive urothelial carcinoma.

Objectives: We aim to evaluate the risk of recurrence after neoadjuvant chemotherapy followed by radical cystectomy, particularly in ypT2 disease in patients with urothelial carcinoma, because it is not clear if all eligible patients with high-risk muscle-invasive urothelial carcinoma should be treated with adjuvant nivolumab. Materials and Methods: We analysed the radiological and clinicopathological features, including cT and ypT stages, of 197 patients who had undergone two to four cycles of cisplatin-based neoadjuvant chemotherapy and radical cystectomy without adjuvant chemotherapy. We stratified the risk of postoperative recurrence by these factors. Results: The median observation period was 29.6 (interquartile range, 11.4-71.7) months, and disease recurrence was observed in 58 patients. Multivariate analysis revealed that ypT stage (P = 0.019) and lymphovascular invasion (P = 0.015) were independent risk factors for postoperative recurrence. The ypT2 group (n = 38) had significantly better recurrence-free survival than the ypT3 group (n = 41) (median recurrence-free survival: not reached vs. 13.4 months, respectively, P = 0.005). In ypT2 disease, the cT2 and ypT2 group (n = 15), which was diagnosed as cT2 preoperatively and then diagnosed as ypT2 postoperatively, had significantly better recurrence-free survival than the cT3/4 and ypT2 group (n = 23) (median recurrence-free survival: not reached vs. 63.1 months, respectively, P = 0.034). There was no significant difference in recurrence-free survival between the ypT ≤ 1 (n = 106) and the cT2 and ypT2 groups (median recurrence-free survival: not reached in both, P = 0.962). Conclusion: Patients with cT2 and ypT2 stage have a relatively low risk of recurrence and thus have a lower need for adjuvant nivolumab, particularly those with ypT2.

The peptide growth factor, phytosulfokine, attenuates pattern-triggered immunity.

Pattern-triggered immunity (PTI) is triggered by recognition of elicitors called microbe-associated molecular patterns (MAMPs). Although immune responses may provide good protection of plants from pathogen attack, excessive immune responses have negative impacts on plant growth and development. Thus, a good balance between positive and negative effects on the immune signaling network is important for plant fitness. However, little information is known about the molecular mechanisms that are involved in attenuation of PTI. Here, we describe a growth-promoting peptide hormone, phytosulfokine (PSK), as attenuating PTI signaling in Arabidopsis. This research was motivated by the observation that expression of the PSK Receptor 1 (PSKR1) gene was induced by MAMP treatment. Plants homozygous for pskr1 T-DNA insertions showed enhanced defense gene expression and seedling growth inhibition triggered by MAMPs. The pskr1 plants also showed enhanced PTI against the bacterial pathogen Pseudomonas syringae. These results indicate that the PSKR-mediated signaling attenuates immune responses. Tyrosyl protein sulfotransferase (TPST) is an enzyme required for production of the mature sulfated PSK. Like pskr1 mutants, a tpst T-DNA insertion line exhibited enhanced MAMP-triggered seedling growth inhibition, which was suppressed by exogenous application of PSK. Thus, PSK signaling mediated by PSKR1 attenuates PTI but stimulates growth.

Electrochemical intercalation of rubidium into graphite, hard carbon, and soft carbon.

The electrochemical insertion of Rb into carbonaceous materials, including graphite, was achieved herein. Rubidium ions were reversibly inserted into and extracted from graphite via electrochemical processes using different non-aqueous electrolytes containing rubidium bis(trifluoromethanesulfonyl)amide (RbTFSA) salts in carbonate esters, glymes, and ionic liquids, similar to the process used for other lighter alkali metal ions such as Li+ and K+. The chemical compositions of the rubidiated graphite were determined to be RbC8, RbC24, and RbC36 at each step of the electrochemical reduction process. Graphite underwent a phase transition to RbC8 exhibiting a stage-1 structure, with stage-3 RbC36 and stage-2 RbC24 as intermediates, as confirmed by ex situ and in situ X-ray diffraction and ex situ Raman spectroscopy, similar to the electrochemical phase evolution of staged potassium graphite intercalation compounds (K-GICs). Furthermore, Rb was reversibly inserted into and extracted from graphitizable and non-graphitizable carbons such as pitch-derived soft carbon and commercial hard carbon, along with other alkali metals such as Li, Na, and K.

Na Diffusion in Hard Carbon Studied with Positive Muon Spin Rotation and Relaxation.

The diffusive nature of Na+ in Na-inserted hard carbon (C x Na), which is the most common anode material for a Na-ion battery, was studied with a positive muon spin rotation and relaxation (µ+SR) technique in transverse, zero, and longitudinal magnetic fields (TF, ZF, and LF) at temperatures between 50 and 375 K, where TF (LF) denotes the applied magnetic field perpendicular (parallel) to the initial muon spin polarization. At temperatures above 150 K, TF-µ+SR measurements showed a distinct motional narrowing behavior, implying that Na+ begins to diffuse above 150 K. The presence of two different muon sites in C x Na was confirmed with ZF- and LF-µ+SR measurements; one is in the Na-inserted graphene layer, and the other is in the Na-vacant graphene layer adjacent to the Na-inserted graphene layer. A systematic increase in the field fluctuation rate (ν) with increasing temperature also evidenced a thermally activated Na diffusion, particularly above 150 K. Assuming the two-dimensional diffusion of Na+ in the graphene layers, the self-diffusion coefficient of Na+ (D Na J) at 300 K was estimated to be 2.5 × 10-11 cm2/s with a thermal activation energy of 39(7) meV.

Case Study: A Japanese patient with metastatic renal cell carcinoma who achieved long-term treatment-free survival with pembrolizumab and axitinib in the KEYNOTE-426 phase III trial of pembrolizumab and axitinib versus sunitinib.

Introduction: Our patient treated with pembrolizumab and axitinib is one of the longest survivors in Japan on KEYNOTE 426, despite adverse events, including delayed-onset hepatitis. We herein present a detailed clinical course and short discussion on the case. Case presentation: This was a 49-year-old male with clear cell renal cell carcinoma and lung metastases. After cytoreductive nephrectomy, treatment with pembrolizumab plus axitinib was initiated and the patient demonstrated a radiographic partial response as best response. The main adverse event was pembrolizumab-induced delayed-onset hepatitis, which was successfully treated with prednisolone. Pembrolizumab was re-initiated and completed. Conclusion: The survival benefit in the present case may be due to the initial potent anti-cancer effects of axitinib and durable immune effects of pembrolizumab, leading to long-term treatment-free survival.

Impact of Cytoreductive Nephrectomy Following Nivolumab Plus Ipilimumab Therapy for Patients With Advanced Renal Cell Carcinoma.

BACKGROUND/AIM: CheckMate 214 study revealed that nivolumab plus ipilimumab combination therapy showed a strong and durable effect compared to sunitinib for patients with advanced renal cell carcinoma (aRCC). Most of the patients underwent previous nephrectomy before systemic treatment. We retrospectively investigated the clinical outcomes of Japanese patients treated with cytoreductive nephrectomy following nivolumab plus ipilimumab for aRCC. PATIENTS AND METHODS: Seventy-nine patients were treated with systemic therapy for aRCC between October 2018 and August 2021 at the Saitama Medical University International Medical Center. Ten of 61 patients treated with nivolumab plus ipilimumab underwent cytoreductive nephrectomy after the combined immunotherapy. RESULTS: The median overall survival and progression-free survival were 24.3 and 15.9 months, respectively. The objective response rate was 50.8%; 9.8% of patients had a complete response, and the median time to objective response was 3.2 (range=1.3-19.7) months. The estimated percentage of patients who sustained an objective response at 30 months was 73.0%. Twenty-three patients (74%) in the complete or partial response (CR/PR) group, 11 patients (52%) in the stable disease (SD) group, and two patients (22%) in the progressive disease (PD) group had immune-related adverse events of grade 3 or higher, respectively. For all 10 patients, cytoreductive nephrectomy following nivolumab plus ipilimumab treatment were completed safely. Three patients achieved a pathological complete response without viable cancer cells. Only two patients had residual lesions on images after deferred cytoreductive nephrectomy; the remaining patients achieved radiological CR. CONCLUSION: Cytoreductive nephrectomy after nivolumab plus ipilimumab treatment could be useful in a limited number of cases, possibly resulting in curative nephrectomy due to the durable therapeutic effect of immunotherapy.

Analysis of glutamate homeostasis by overexpression of Fd-GOGAT gene in Arabidopsis thaliana.

Glutamate plays a central role in nitrogen flow and serves as a nitrogen donor for the production of amino acids. In plants, some amino acids work as buffers: during photorespiration, ammonium derived from the conversion of glycine to serine is promptly reassimilated into glutamate by the glutamine synthetase (GS-2)/ferredoxin-dependent glutamate synthase (Fd-GOGAT) cycle. The glutamate concentration is relatively stable compared with those of other amino acids under environmental changes. The few studies dealing with glutamate homeostasis have but all used knockouts or mutants of these enzymes. Here, we generated Fd-GOGAT (GLU1)-overexpressing Arabidopsis plants to analyze changes in the amino acid pool caused by glutamate overproduction under different ammonium conditions controlled by CO(2) concentration, light intensity and nitrate concentration. Under photorespiratory conditions with sufficient ammonium supply, aspartate increased and glutamine and glycine decreased, but glutamate barely changed. Under non-photorespiratory conditions, however, glutamate and most other amino acids increased. These results suggest that the synthesized glutamate is promptly converted into other amino acids, especially aspartate. In addition, ammonium supply by photorespiration does not limit glutamate biosynthesis, but glutamine and glycine are important. This study will contribute to the understanding of glutamate homeostasis in plants.

Asymmetric hydrogenation of aryl ketones mediated by a copper catalyst.

[reaction: see text] A novel asymmetric hydrogenation protocol using a copper catalyst is reported. A Cu(I) complex in the presence of nonracemic BDPP hydrogenates aryl ketones with moderate to high enantioselectivity.

Peptidoglycan from fermentation by-product triggers defense responses in grapevine.

Plants are constantly under attack from a variety of microorganisms, and rely on a series of complex detection and response systems to protect themselves from infection. Here, we found that a by-product of glutamate fermentation triggered defense responses in grapevine, increasing the expression of defense response genes in cultured cells, foliar chitinase activity, and resistance to infection by downy mildew in leaf explants. To identify the molecule that triggered this innate immunity, we fractionated and purified candidates extracted from Corynebacterium glutamicum, a bacterium used in the production of amino acids by fermentation. Using hydrolysis by lysozyme, a silkworm larva plasma detection system, and gel filtration analysis, we identified peptidoglycan as inducing the defense responses. Peptidoglycans of Escherichia coli, Bacillus subtilis, and Staphylococcus aureus also generated similar defensive responses.

Mechanisms underlying robustness and tunability in a plant immune signaling network.

The plant immune signaling network needs to be robust against attack from fast-evolving pathogens and tunable to optimize immune responses. We investigated the basis of robustness and tunability in the signaling network controlling pattern-triggered immunity (PTI) in Arabidopsis. A dynamic network model containing four major signaling sectors, the jasmonate, ethylene, phytoalexin-deficient 4, and salicylate sectors, which together govern up to 80% of the PTI levels, was built using data for dynamic sector activities and PTI levels under exhaustive combinatorial sector perturbations. Our regularized multiple regression model had a high level of predictive power and captured known and unexpected signal flows in the network. The sole inhibitory sector in the model, the ethylene sector, contributed centrally to network robustness via its inhibition of the jasmonate sector. The model's multiple input sites linked specific signal input patterns varying in strength and timing to different network response patterns, indicating a mechanism enabling tunability.

Pattern-triggered immunity suppresses programmed cell death triggered by fumonisin b1.

Programmed cell death (PCD) is a crucial process for plant innate immunity and development. In plant innate immunity, PCD is believed to prevent the spread of pathogens from the infection site. Although proper control of PCD is important for plant fitness, we have limited understanding of the molecular mechanisms regulating plant PCD. Plant innate immunity triggered by recognition of effectors (effector-triggered immunity, ETI) is often associated with PCD. However pattern-triggered immunity (PTI), which is triggered by recognition of elicitors called microbe-associated molecular patterns (MAMPs), is not. Therefore we hypothesized that PTI might suppress PCD. Here we report that PCD triggered by the mycotoxin fumonisin B1 (FB1) can be suppressed by PTI in Arabidopsis. FB1-triggered cell death was suppressed by treatment with the MAMPs flg22 (a part of bacterial flagellin) or elf18 (a part of the bacterial elongation factor EF-Tu) but not chitin (a component of fungal cell walls). Although plant hormone signaling is associated with PCD and PTI, both FB1-triggered cell death and suppression of cell death by flg22 treatment were still observed in mutants deficient in jasmonic acid (JA), ethylene (ET) and salicylic acid (SA) signaling. The MAP kinases MPK3 and MPK6 are transiently activated and inactivated within one hour during PTI. We found that FB1 activated MPK3 and MPK6 about 36-48 hours after treatment. Interestingly, this late activation was attenuated by flg22 treatment. These results suggest that PTI suppression of FB1-triggered cell death may involve suppression of MPK3/MPK6 signaling but does not require JA/ET/SA signaling.

Reproductive organs regulate leaf nitrogen metabolism mediated by cytokinin signal.

The metabolism of vegetative organs in plants changes during the development of the reproductive organs. The regulation of this metabolism is important in the control of crop productivity. However, the complexity of the regulatory systems makes it difficult to elucidate their mechanisms. To examine these mechanisms, we constructed model experiments using Arabidopsis to analyze metabolic and gene expression changes during leaf-stage progression and after removal of the reproductive organs. Leaf gene expression levels and content of major amino acids, both of which decreased during leaf-stage progression, increased after removal of the reproductive organs. In particular, the levels of expression of cytokinin biosynthesis genes and cytokinin-responsive genes and the cytokinin content increased after removal of the reproductive organs. Analysis of plants with knockout of a cytokinin-biosynthetic gene (AtIPT3) and a cytokinin receptor gene (AHK3) indicated that glutamate dehydrogenase genes (GDH3) were regulated by cytokinin signaling. These data suggest that cytokinins regulate communication between reproductive and vegetative organs, and that GDH3 is one target of the cytokinin-mediated regulation of nitrogen metabolism.

Glutamate:glyoxylate aminotransferase modulates amino acid content during photorespiration.

In photorespiration, peroxisomal glutamate:glyoxylate aminotransferase (GGAT) catalyzes the reaction of glutamate and glyoxylate to produce 2-oxoglutarate and glycine. Previous studies demonstrated that alanine aminotransferase-like protein functions as a photorespiratory GGAT. Photorespiratory transamination to glyoxylate, which is mediated by GGAT and serine glyoxylate aminotransferase (SGAT), is believed to play an important role in the biosynthesis and metabolism of major amino acids. To better understand its role in the regulation of amino acid levels, we produced 42 GGAT1 overexpression lines that express different levels of GGAT1 mRNA. The levels of free serine, glycine, and citrulline increased markedly in GGAT1 overexpression lines compared with levels in the wild type, and levels of these amino acids were strongly correlated with levels of GGAT1 mRNA and GGAT activity in the leaves. This accumulation began soon after exposure to light and was repressed under high levels of CO(2). Light and nutrient conditions both affected the amino acid profiles; supplementation with NH(4)NO(3) increased the levels of some amino acids compared with the controls. The results suggest that the photorespiratory aminotransferase reactions catalyzed by GGAT and SGAT are both important regulators of amino acid content.

Identification of photorespiratory glutamate:glyoxylate aminotransferase (GGAT) gene in Arabidopsis.

In the photorespiratory process, peroxisomal glutamate:glyoxylate aminotransferase (GGAT) catalyzes the reaction of glutamate and glyoxylate to 2-oxoglutarate and glycine. Although GGAT has been assumed to play important roles for the transamination in photorespiratory carbon cycles, the gene encoding GGAT has not been identified. Here, we report that an alanine:2-oxoglutarate aminotransferase (AOAT)-like protein functions as GGAT in peroxisomes. Arabidopsis has four genes encoding AOAT-like proteins and two of them (namely AOAT1 and AOAT2) contain peroxisomal targeting signal 1 (PTS1). The expression analysis of mRNA encoding AOATs and EST information suggested that AOAT1 was the major protein in green leaves. When AOAT1 fused to green fluorescent protein (GFP) was expressed in BY-2 cells, it was found to be localized to peroxisomes depending on PTS1. By screening of Arabidopsis T-DNA insertion lines, an AOAT1 knockout line (aoat1-1) was isolated. The activity of GGAT and alanine:glyoxylate aminotransferase (AGAT) in the above-ground tissues of aoat1-1 was reduced drastically and, AOAT and glutamate:pyruvate aminotransferase (GPAT) activity also decreased. Peroxisomal GGAT was detected in the wild type but not in aoat1-1. The growth rate was repressed in aoat1-1 grown under high irradiation or without sugar, though differences were slight in aoat1-1 grown under low irradiation, high-CO2 (0.3%) or high-sugar (3% sucrose) conditions. These phenotypes resembled those of photorespiration-deficient mutants. Glutamate levels increased and serine levels decreased in aoat1-1 grown in normal air conditions. Based on these results, it was concluded that AOAT1 is targeted to peroxisomes, functions as a photorespiratory GGAT, plays a markedly important role for plant growth and the metabolism of amino acids.