Nonpeptidergic neurons suppress mast cells via glutamate to maintain skin homeostasis.

Cutaneous mast cells mediate numerous skin inflammatory processes and have anatomical and functional associations with sensory afferent neurons. We reveal that epidermal nerve endings from a subset of sensory nonpeptidergic neurons expressing MrgprD are reduced by the absence of Langerhans cells. Loss of epidermal innervation or ablation of MrgprD-expressing neurons increased expression of a mast cell gene module, including the activating receptor, Mrgprb2, resulting in increased mast cell degranulation and cutaneous inflammation in multiple disease models. Agonism of MrgprD-expressing neurons reduced expression of module genes and suppressed mast cell responses. MrgprD-expressing neurons released glutamate which was increased by MrgprD agonism. Inhibiting glutamate release or glutamate receptor binding yielded hyperresponsive mast cells with a genomic state similar to that in mice lacking MrgprD-expressing neurons. These data demonstrate that MrgprD-expressing neurons suppress mast cell hyperresponsiveness and skin inflammation via glutamate release, thereby revealing an unexpected neuroimmune mechanism maintaining cutaneous immune homeostasis.

Trapping biosynthetic acyl-enzyme intermediates with encoded 2,3-diaminopropionic acid.

Many enzymes catalyse reactions that proceed through covalent acyl-enzyme (ester or thioester) intermediates1. These enzymes include serine hydrolases2,3 (encoded by one per cent of human genes, and including serine proteases and thioesterases), cysteine proteases (including caspases), and many components of the ubiquitination machinery4,5. Their important acyl-enzyme intermediates are unstable, commonly having half-lives of minutes to hours6. In some cases, acyl-enzyme complexes can be stabilized using substrate analogues or active-site mutations but, although these approaches can provide valuable insight7-10, they often result in complexes that are substantially non-native. Here we develop a strategy for incorporating 2,3-diaminopropionic acid (DAP) into recombinant proteins, via expansion of the genetic code11. We show that replacing catalytic cysteine or serine residues of enzymes with DAP permits their first-step reaction with native substrates, allowing the efficient capture of acyl-enzyme complexes that are linked through a stable amide bond. For one of these enzymes, the thioesterase domain of valinomycin synthetase12, we elucidate the biosynthetic pathway by which it progressively oligomerizes tetradepsipeptidyl substrates to a dodecadepsipeptidyl intermediate, which it then cyclizes to produce valinomycin. By trapping the first and last acyl-thioesterase intermediates in the catalytic cycle as DAP conjugates, we provide structural insight into how conformational changes in thioesterase domains of such nonribosomal peptide synthetases control the oligomerization and cyclization of linear substrates. The encoding of DAP will facilitate the characterization of diverse acyl-enzyme complexes, and may be extended to capturing the native substrates of transiently acylated proteins of unknown function.

Coenzyme biosynthesis in response to precursor availability reveals incorporation of ß-alanine from pantothenate in prototrophic bacteria.

Coenzymes are important for all classes of enzymatic reactions and essential for cellular metabolism. Most coenzymes are synthesized from dedicated precursors, also referred to as vitamins, which prototrophic bacteria can either produce themselves from simpler substrates or take up from the environment. The extent to which prototrophs use supplied vitamins and whether externally available vitamins affect the size of intracellular coenzyme pools and control endogenous vitamin synthesis is currently largely unknown. Here, we studied coenzyme pool sizes and vitamin incorporation into coenzymes during growth on different carbon sources and vitamin supplementation regimes using metabolomics approaches. We found that the model bacterium Escherichia coli incorporated pyridoxal, niacin, and pantothenate into pyridoxal 5'-phosphate, NAD, and coenzyme A (CoA), respectively. In contrast, riboflavin was not taken up and was produced exclusively endogenously. Coenzyme pools were mostly homeostatic and not affected by externally supplied precursors. Remarkably, we found that pantothenate is not incorporated into CoA as such but is first degraded to pantoate and ß-alanine and then rebuilt. This pattern was conserved in various bacterial isolates, suggesting a preference for ß-alanine over pantothenate utilization in CoA synthesis. Finally, we found that the endogenous synthesis of coenzyme precursors remains active when vitamins are supplied, which is consistent with described expression data of genes for enzymes involved in coenzyme biosynthesis under these conditions. Continued production of endogenous coenzymes may ensure rapid synthesis of the mature coenzyme under changing environmental conditions, protect against coenzyme limitation, and explain vitamin availability in naturally oligotrophic environments.

How an overlooked gene in coenzyme a synthesis solved an enzyme mechanism predicament.

Coenzyme A (CoA) is an essential cofactor throughout biology. The first committed step in the CoA synthetic pathway is synthesis of ß-alanine from aspartate. In Escherichia coli and Salmonella enterica panD encodes the responsible enzyme, aspartate-1-decarboxylase, as a proenzyme. To become active, the E. coli and S. enterica PanD proenzymes must undergo an autocatalytic cleavage to form the pyruvyl cofactor that catalyzes decarboxylation. A problem was that the autocatalytic cleavage was too slow to support growth. A long-neglected gene (now called panZ) was belatedly found to encode the protein that increases autocatalytic cleavage of the PanD proenzyme to a physiologically relevant rate. PanZ must bind CoA or acetyl-CoA to interact with the PanD proenzyme and accelerate cleavage. The CoA/acetyl-CoA dependence has led to proposals that the PanD-PanZ CoA/acetyl-CoA interaction regulates CoA synthesis. Unfortunately, regulation of ß-alanine synthesis is very weak or absent. However, the PanD-PanZ interaction provides an explanation for the toxicity of the CoA anti-metabolite, N5-pentyl pantothenamide.

Fine and combinatorial regulation of key metabolic pathway for enhanced ß-alanine biosynthesis with non-inducible Escherichia coli.

ß-Alanine is the only ß-amino acid in nature and one of the most important three-carbon chemicals. This work was aimed to construct a non-inducible ß-alanine producer with enhanced metabolic flux towards ß-alanine biosynthesis in Escherichia coli. First of all, the assembled E. coli endogenous promoters and 5'-untranslated regions (PUTR) were screened to finely regulate the combinatorial expression of genes panDBS and aspBCG for an optimal flux match between two key pathways. Subsequently, additional copies of key genes (panDBS K104S and ppc) were chromosomally introduced into the host A1. On these bases, dynamical regulation of the gene thrA was performed to reduce the carbon flux directed in the competitive pathway. Finally, the ß-alanine titer reached 10.25 g/L by strain A14-R15, 361.7% higher than that of the original strain. Under fed-batch fermentation in a 5-L fermentor, a titer of 57.13 g/L ß-alanine was achieved at 80 h. This is the highest titer of ß-alanine production ever reported using non-inducible engineered E. coli. This metabolic modification strategy for optimal carbon flux distribution developed in this work could also be used for the production of various metabolic products.

Effect of amino acid enriched diets on hemolymph amino acid composition in honey bees.

Amino acids (AAs) are an abundant class of nectar solutes, and they are involved in the nectar attractiveness to flower visitors. Among the various AAs, proline is the most abundant proteogenic AA, and γ-amino butyric acid (GABA) and ß-alanine are the two most abundant non-proteogenic AAs. These three AAs are known to affect insect physiology, being involved in flight metabolism and neurotransmission. The aim of this study was to investigate the effects of artificial diets enriched with either ß-alanine, GABA, or proline on consumption, survival, and hemolymph composition in honey bees belonging to two different ages and with different metabolism (i.e., newly emerged and foragers). Differences in feed intake among diets were not observed, while a diet enriched with ß-alanine improved the survival rate of newly emerged honey bees compared to the control group. Variations in the hemolymph AA concentrations occurred only in newly emerged honey bees, according to the diet and the time of hemolymph sampling. A greater susceptibility of young honey bees to enriched diets than older honey bees was observed. The variations in the concentrations of hemolymph AAs reflect either the accumulation of dietary AAs or the existence of metabolic pathways that may lead to the conversion of dietary AAs into different ones. This investigation could be an initial contribution to studying the complex dynamics that regulate hemolymph AA composition and its effect on honey bee physiology.

Reposition: Focalizing ß-Alanine Metabolism and the Anti-Inflammatory Effects of Its Metabolite Based on Multi-Omics Datasets.

The incorporation of multi-omics data methodologies facilitates the concurrent examination of proteins, metabolites, and genes associated with inflammation, thereby leveraging multi-dimensional biological data to achieve a comprehensive understanding of the complexities involved in the progression of inflammation. Inspired by ensemble learning principles, we implemented ID normalization preprocessing, categorical sampling homogenization, and pathway enrichment across each sample matrix derived from multi-omics datasets available in the literature, directing our focus on inflammation-related targets within lipopolysaccharide (LPS)-stimulated RAW264.7 cells towards ß-alanine metabolism. Additionally, through the use of LPS-treated RAW264.7 cells, we tentatively validated the anti-inflammatory properties of the metabolite Ureidopropionic acid, originating from ß-alanine metabolism, by evaluating cell viability, nitric oxide production levels, and mRNA expression of inflammatory biomarkers. In conclusion, our research represents the first instance of an integrated analysis of multi-omics datasets pertaining to LPS-stimulated RAW264.7 cells as documented in the literature, underscoring the pivotal role of ß-alanine metabolism in cellular inflammation and successfully identifying Ureidopropionic acid as a novel anti-inflammatory compound. Moreover, the findings from database predictions and molecular docking studies indicated that the inflammatory-related pathways and proteins may serve as potential mechanistic targets for Ureidopropionic acid.

Characterization of a novel ß-alanine biosynthetic pathway consisting of promiscuous metabolic enzymes.

Bacteria adapt to utilize the nutrients available in their environment through a sophisticated metabolic system composed of highly specialized enzymes. Although these enzymes can metabolize molecules other than those for which they evolved, their efficiency toward promiscuous substrates is considered too low to be of physiological relevance. Herein, we investigated the possibility that these promiscuous enzymes are actually efficient enough at metabolizing secondary substrates to modify the phenotype of the cell. For example, in the bacterium Acinetobacter baylyi ADP1 (ADP1), panD (coding for l-aspartate decarboxylase) encodes the only protein known to catalyze the synthesis of ß-alanine, an obligate intermediate in CoA synthesis. However, we show that the ADP1 ΔpanD mutant could also form this molecule through an unknown metabolic pathway arising from promiscuous enzymes and grow as efficiently as the wildtype strain. Using metabolomic analyses, we identified 1,3-diaminopropane and 3-aminopropanal as intermediates in this novel pathway. We also conducted activity screening and enzyme kinetics to elucidate candidate enzymes involved in this pathway, including 2,4-diaminobutyrate aminotransferase (Dat) and 2,4-diaminobutyrate decarboxylase (Ddc) and validated this pathway in vivo by analyzing the phenotype of mutant bacterial strains. Finally, we experimentally demonstrate that this novel metabolic route is not restricted to ADP1. We propose that the occurrence of conserved genes in hundreds of genomes across many phyla suggests that this previously undescribed pathway is widespread in prokaryotes.

Screening and identification of genes involved in ß-alanine biosynthesis in Bacillus subtilis.

ß-alanine is the only naturally occurring ß-amino acid, which is widely used in medicine, food, and feed fields, and generally produced through synthetic biological methods based on engineered strains of Escherichia coli or Corynebacterium glutamicum. However, the ß-alanine biosynthesis in Bacillus subtilis, a traditional industrial model microorganism of food safety grade, has not been thoroughly explored. In this study, the native l-aspartate-α-decarboxylase was overexpressed in B. subtilis 168 to obtain an increase of 842% in ß-alanine production. A total of 16 single-gene knockout strains were constructed to block the competitive consumption pathways to identify a total of 6 genes (i.e., ptsG, fbp, ydaP, yhfS, mmgA, and pckA) involved in ß-alanine synthesis, while the multigene knockout of these 6 genes obtained an increased ß-alanine production by 40.1%. Ten single-gene suppression strains with the competitive metabolic pathways inhibited revealed that the inhibited expressions of genes glmS, accB, and accA enhanced the ß-alanine production. The introduction of heterologous phosphoenolpyruvate carboxylase increased the ß-alanine production by 81.7%, which was 17-fold higher than that of the original strain. This was the first study using multiple molecular strategies to investigate the biosynthetic pathway of ß-alanine in B. subtilis and to identify the genetic factors limiting the excessive synthesis of ß-alanine by microorganisms.

Carnosine increases insulin-stimulated glucose uptake and reduces methylglyoxal-modified proteins in type-2 diabetic human skeletal muscle cells.

Type-2 diabetes (T2D) is characterised by a dysregulation of metabolism, including skeletal muscle insulin resistance, mitochondrial dysfunction, and oxidative stress. Reactive species, such as methylglyoxal (MGO) and 4-hydroxynonenal (4-HNE), positively associate with T2D disease severity and can directly interfere with insulin signalling and glucose uptake in skeletal muscle by modifying cellular proteins. The multifunctional dipeptide carnosine, and its rate-limiting precursor ß-alanine, have recently been shown to improve glycaemic control in humans and rodents with diabetes. However, the precise mechanisms are unclear and research in human skeletal muscle is limited. Herein, we present novel findings in primary human T2D and lean healthy control (LHC) skeletal muscle cells. Cells were differentiated to myotubes, and treated with 10 mM carnosine, 10 mM ß-alanine, or control for 4-days. T2D cells had reduced ATP-linked and maximal respiration compared with LHC cells (p = 0.016 and p = 0.005). Treatment with 10 mM carnosine significantly increased insulin-stimulated glucose uptake in T2D cells (p = 0.047); with no effect in LHC cells. Insulin-stimulation increased MGO-modified proteins in T2D cells by 47%; treatment with carnosine attenuated this increase to 9.7% (p = 0.011). There was no effect treatment on cell viability or expression of other proteins. These findings suggest that the beneficial effects of carnosine on glycaemic control may be explained by its scavenging actions in human skeletal muscle.

Development of probiotic E. coli Nissle 1917 for ß-alanine production by using protein and metabolic engineering.

ß-alanine has been used in food and pharmaceutical industries. Although Escherichia coli Nissle 1917 (EcN) is generally considered safe and engineered as living therapeutics, engineering EcN for producing industrial metabolites has rarely been explored. Here, by protein and metabolic engineering, EcN was engineered for producing ß-alanine from glucose. First, an aspartate-α-decarboxylase variant ADCK43Y with improved activity was identified and over-expressed in EcN, promoting the titer of ß-alanine from an undetectable level to 0.46 g/L. Second, directing the metabolic flux towards L-aspartate increased the titer of ß-alanine to 0.92 g/L. Third, the yield of ß-alanine was elevated to 1.19 g/L by blocking conversion of phosphoenolpyruvate to pyruvate, and further increased to 2.14 g/L through optimizing culture medium. Finally, the engineered EcN produced 11.9 g/L ß-alanine in fed-batch fermentation. Our work not only shows the potential of EcN as a valuable industrial platform, but also facilitates production of ß-alanine via fermentation. KEY POINTS: • Escherichia coli Nissle 1917 (EcN) was engineered as a ß-alanine producing cell factory • Identification of a decarboxylase variant ADCK43Y with improved activity • Directing the metabolic flux to L-ASP and expressing ADCK43Y elevated the titer of ß-alanine to 11.9 g/L.

Engineering Escherichia coli to assimilate ß-alanine as a major carbon source.

The threat of global plastic waste accumulation has spurred the exploration of plastics derived from biological sources. A well-known example is polyester made of 1,3-propanediol (1,3-PDO). However, there is no known pathway to assimilate 1,3-PDO into the central carbon metabolism, posing a potential challenge to upcycling such plastic wastes. Here, we proposed that the 1,3-PDO assimilation pathway could pass through malonate semialdehyde (MSA) as an intermediate. Since MSA is a toxic aldehyde, ß-alanine was chosen as a surrogate substrate in this study to construct the lower part of the proposed pathway. To this end, we successfully engineered E. coli MG1655 to assimilate ß-alanine as the major carbon source. ß-alanine could be easily converted into MSA using a ß-alanine/pyruvate transaminase from Pseudomonas aeruginosa (PaBapt). However, the subsequent step to generate acetyl-CoA from MSA was unknown. After a series of phenotype screenings, adaptive laboratory evolution and transcriptomic analysis, two CoA-acylating MSA dehydrogenases from Vibrio natriegens (VnMmsD), were found to be able to complete the metabolic pathway. Optical density at 600 nm (OD600) of the resulting strain E. coli BA02 could reach 4.5 after 96 h. Two approaches were subsequently used to improve its performance. First, PaBapt and both VnMmsDs were expressed from a single plasmid to mitigate antibiotic stress. Second, a native 3-hydroxy acid dehydrogenase (EcYdfG) was disrupted to address the carbon loss to 3-hydroxypropionate (3-HP) production from MSA. OD600 of the best-performing strain E. coli BA07∆ could reach 6 within 24 h using 5 g/L ß-alanine. The construction of E. coli BA07∆ lays a solid foundation to establishing a 1,3-PDO assimilation pathway. KEYPOINTS: • This study demonstrates the implementation of a metabolic pathway to assimilate ß-alanine as the major carbon source in E. coli MG1655. • Two V. natriegens CoA-acylating methyl malonate semialdehyde dehydrogenases were used to complete the pathway in E. coli BA02. • The construction of E. coli BA02 also revealed the plasmid fusion event between two plasmids with the same replication origin.

Effects of dietary ß-alanine supplementation on growth performance, meat quality, carnosine content, amino acid composition and muscular antioxidant capacity in Chinese indigenous Ningxiang pig.

ß-alanine has been demonstrated to improve carcass traits and meat quality of animals. However, no research has been found on the effects of dietary ß-alanine in the meat quality control of finishing pigs, which are among the research focus. Therefore, this study aimed to evaluate the effects of dietary ß-alanine supplementation on growth performance, meat quality, carnosine content, amino acid composition and muscular antioxidant capacity of Chinese indigenous Ningxiang pigs. The treatments contained a basal diet (control, CON) and a basal diet supplemented with 600 mg/kg ß-alanine. Each treatment group consisted of five pens, with five pigs per pen. Results showed that compared with CON, supplemental ß-alanine did not affect the final body weight, average daily gain, average daily feed intake and the feed-to-gain ratio of pigs. Dietary ß-alanine supplementation tended to increase the pH45 min (p = 0.071) while decreasing the shear force (p = 0.085) and the drip loss (p = 0.091). Moreover, it improved (p < 0.05) the activities of glutathione peroxidase and catalase and lessened (p < 0.05) malondialdehyde concentration. Added ß-alanine in diets of finishing pigs could enhance the concentrations of arginine, alanine, and glutamate (p < 0.05) in the longissimus dorsi muscle and tended to raise the levels of cysteine, glycine and anserine (p = 0.060, p = 0.098 and p = 0.091 respectively). Taken together, our results showed that dietary ß-alanine supplementation contributed to the improvement of the carcass traits, meat quality and anserine content, the amelioration of muscle antioxidant capacity and the regulation of amino acid composition in Chinese indigenous Ningxiang pigs.

Positive interaction between GPER and ß-alanine in the dorsal root ganglion uncovers potential mechanisms: mediating continuous neuronal sensitization and neuroinflammation responses in neuropathic pain.

BACKGROUND: The pathogenesis of neuropathic pain and the reasons for the prolonged unhealing remain unknown. Increasing evidence suggests that sex oestrogen differences play a role in pain sensitivity, but few studies have focused on the oestrogen receptor which may be an important molecular component contributing to peripheral pain transduction. We aimed to investigate the impact of oestrogen receptors on the nociceptive neuronal response in the dorsal root ganglion (DRG) and spinal dorsal horn using a spared nerve injury (SNI) rat model of chronic pain. METHODS: We intrathecally (i.t.) administered a class of oestrogen receptor antagonists and agonists intrathecal (i.t.) administrated to male rats with SNI or normal rats to identify the main receptor. Moreover, we assessed genes identified through genomic metabolic analysis to determine the key metabolism point and elucidate potential mechanisms mediating continuous neuronal sensitization and neuroinflammatory responses in neuropathic pain. The excitability of DRG neurons was detected using the patch-clamp technique. Primary culture was used to extract microglia and DRG neurons, and siRNA transfection was used to silence receptor protein expression. Immunofluorescence, Western blotting, RT-PCR and behavioural testing were used to assess the expression, cellular distribution, and actions of the main receptor and its related signalling molecules. RESULTS: Increasing the expression and function of G protein-coupled oestrogen receptor (GPER), but not oestrogen receptor-α (ERα) and oestrogen receptor-ß (ERß), in the DRG neuron and microglia, but not the dorsal spinal cord, contributed to SNI-induced neuronal sensitization. Inhibiting GPER expression in the DRG alleviated SNI-induced pain behaviours and neuroinflammation by simultaneously downregulating iNOS, IL-1ß and IL-6 expression and restoring GABAα2 expression. Additionally, the positive interaction between GPER and ß-alanine and subsequent ß-alanine accumulation enhances pain sensation and promotes chronic pain development. CONCLUSION: GPER activation in the DRG induces a positive association between ß-alanine with iNOS, IL-1ß and IL-6 expression and represses GABAα2 involved in post-SNI neuropathic pain development. Blocking GPER and eliminating ß-alanine in the DRG neurons and microglia may prevent neuropathic pain development.

Systems metabolic engineering of Corynebacterium glutamicum for the efficient production of ß-alanine.

ß-Alanine is an important ß-amino acid with a growing demand in a wide range of applications in chemical and food industries. However, current industrial production of ß-alanine relies on chemical synthesis, which usually involves harmful raw materials and harsh production conditions. Thus, there has been increasing demand for more sustainable, yet efficient production process of ß-alanine. In this study, we constructed Corynebacterium glutamicum strains for the highly efficient production of ß-alanine through systems metabolic engineering. First, aspartate 1-decarboxylases (ADCs) from seven different bacteria were screened, and the Bacillus subtilis ADC showing the most efficient ß-alanine biosynthesis was used to construct a ß-alanine-producing base strain. Next, genome-scale metabolic simulations were conducted to optimize multiple metabolic pathways in the base strain, including phosphotransferase system (PTS)-independent glucose uptake system and the biosynthesis of key precursors, including oxaloacetate and L-aspartate. TCA cycle was further engineered for the streamlined supply of key precursors. Finally, a putative ß-alanine exporter was newly identified, and its overexpression further improved the ß-alanine production. Fed-batch fermentation of the final engineered strain BAL10 (pBA2_tr18) produced 166.6 g/L of ß-alanine with the yield and productivity of 0.28 g/g glucose and 1.74 g/L/h, respectively. To our knowledge, this production performance corresponds to the highest titer, yield and productivity reported to date for the microbial fermentation.

Metabolic engineering of E. coli for ß-alanine production using a multi-biosensor enabled approach.

ß-alanine is an important biomolecule used in nutraceuticals, pharmaceuticals, and chemical synthesis. The relatively eco-friendly bioproduction of ß-alanine has recently attracted more interest than petroleum-based chemical synthesis. In this work, we developed two types of in vivo high-throughput screening platforms, wherein one was utilized to identify a novel target ribonuclease E (encoded by rne) as well as a redox-cofactor balancing module that can enhance de novo ß-alanine biosynthesis from glucose, and the other was employed for screening fermentation conditions. When combining these approaches with rational upstream and downstream module engineering, an engineered E. coli producer was developed that exhibited 3.4- and 6.6-fold improvement in ß-alanine yield (0.85 mol ß-alanine/mole glucose) and specific ß-alanine production (0.74 g/L/OD600), respectively, compared to the parental strain in a minimal medium. Across all of the strains constructed, the best yielding strain exhibited 1.08 mol ß-alanine/mole glucose (equivalent to 81.2% of theoretic yield). The final engineered strain produced 6.98 g/L ß-alanine in a batch-mode bioreactor and 34.8 g/L through a whole-cell catalysis. This approach demonstrates the utility of biosensor-enabled high-throughput screening for the production of ß-alanine.

Taurine and its transporter TAUT positively affect male reproduction and early embryo development.

STUDY QUESTION: Are taurine and its transporter TAUT associated with spermiogenesis and early embryo development? SUMMARY ANSWER: Morphologically abnormal spermatozoa increased after local functional interference by intratesticular injection, and taurine depletion significantly reduced the normal embryo numbers in vivo and blastocyst formation rate in vitro. WHAT IS KNOWN ALREADY: Taurine is one of the most abundant amino acids in the male reproductive system and it has been demonstrated that taurine can efficiently improve spermatogenic function in rat models of testicular injury. However, limited information is known about the role of taurine and its transporter TAUT in spermatogenesis and early embryo development. STUDY DESIGN, SIZE, DURATION: Clinical characteristics from 110 couples who have experienced recurrent pregnancy loss (RPL) were collected from December 2014 to March 2018. According to whether a fetal heartbeat was seen in the previous pregnancy under ultrasonic monitoring, patients with RPL were divided into two groups: an RPL without heartbeat (pregnancy with no fetal heartbeat, ROH) group, and an RPL with heartbeat (one or more pregnancies with fetal heartbeat, RWH) group. Semen samples (21 ROH and 20 RWH) were finally used for metabolomic analysis. Furthermore, semen samples were obtained from 30 patients with teratozoospermia (normal sperm morphology <4%) seeking evaluation for infertility and 25 age-matched control subjects with normal semen quality for western blotting. Animal experiments were performed in CD-1/ICR mice. PARTICIPANTS/MATERIALS, SETTING, METHODS: Metabolomics was performed to determine the metabolic changes between the ROH and RWH groups. Sperm proteins from patients with teratozoospermia and healthy controls were extracted for detecting TAUT expression using western blot analysis. Immunofluorescence was used to characterize the localization of TAUT in the testis and ejaculated spermatozoa. Functional analysis in mice was performed by intratesticular injection of siRNAs or antagonist (ß-alanine) and 5% ß-alanine was provided in drinking water to 3-week-old male mice for 5 weeks with the aim of depleting taurine. Murine epididymal spermatozoa were stained with hematoxylin and eosin for morphological assessment. IVF and mating tests were performed in mice for assessing fertility. MAIN RESULTS AND THE ROLE OF CHANCE: Metabolomic analysis demonstrated that the taurine content was lower in spermatozoa but higher in seminal plasma from the ROH than the RWH group. TAUT expression was lower in spermatozoa from patients with teratozoospermia than controls. Immunofluorescence showed that TAUT was localized to the manchette in mouse elongated spermatids functional analysis showed that morphologically abnormal spermatozoa increased after interference, and this defect increased after supplementation with 5% ß-alanine but was improved by 5% taurine supplementation. Supplementation with 5% ß-alanine significantly reduced the normal embryo number in the mouse uterus as well as blastocyst formation rate in vitro. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The sample size was low and larger cohorts are needed to confirm the positive effect of taurine on human sperm quality. A comprehensive safety examination should be performed to evaluate whether taurine is a possible treatment for teratozoospermia. Furthermore, the specific molecular mechanism of TAUT involvement in spermiogenesis remains to be clarified. WIDER IMPLICATIONS OF THE FINDINGS: The study provides new insights into the role of taurine and its transporter TAUT in male reproduction and embryo development. The results also indicate that TAUT is a promising molecular candidate for the assessment of sperm quality, which may contribute to the diagnosis and treatment for teratozoospermia. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from the National Natural Science Foundation of China (no. 81774075, 31900605, 81971451), Jiangsu Science and Technology Program Grant (BK20190654) and Maternal and child health scientific research of Jiangsu Province (F202121). The authors declare no competing financial interests.

Stimulation of uncoupling protein 1 expression by ß-alanine in brown adipocytes.

Carnosine, which is abundant in meat, is a dipeptide composed of ß-alanine and histidine, known to afford various health benefits. It has been suggested that carnosine can elicit an anti-obesity effect via induction and activation of brown/beige adipocytes responsible for non-shivering thermogenesis. However, the relationship between carnosine and brown/beige adipocytes has not been comprehensively elucidated. We hypothesized that ß-alanine directly modulates brown/beige adipogenesis and performed an in vitro assessment to test this hypothesis. HB2 brown preadipocytes were differentiated using insulin from day 0. Cells were treated with various concentrations of ß-alanine (12.5-100 µM) during adipogenesis (days 0-8) and differentiation (days 8-10). Then, cells were further stimulated with or without forskolin, an activator of the cAMP-dependent protein kinase pathway, on day 8 or day 10 for 4 h before harvesting. We observed that HB2 cells expressed molecules related to the transport and signal transduction of ß-alanine. Treatment with ß-alanine during brown adipogenesis dose-dependently enhanced forskolin-induced Ucp1 expression; this was not observed in differentiated brown adipocytes. Consistent with these findings, treatment with ß-alanine during days 0-8 increased phosphorylation levels of CREB in forskolin-treated HB2 cells. In addition, ß-alanine treatment during brown adipogenesis increased the expression of Pparα, known to induce brown/beige adipogenesis, in a dose-dependent manner. These findings revealed that ß-alanine could target HB2 adipogenic cells and enhance forskolin-induced Ucp1 expression during brown adipogenesis, possibly by accelerating phosphorylation and activation of CREB. Thus, ß-alanine, a carnosine-constituting amino acid, might directly act on brown adipogenic cells to stimulate energy expenditure.

Extra-helical binding site of a glucagon receptor antagonist.

Glucagon is a 29-amino-acid peptide released from the α-cells of the islet of Langerhans, which has a key role in glucose homeostasis. Glucagon action is transduced by the class B G-protein-coupled glucagon receptor (GCGR), which is located on liver, kidney, intestinal smooth muscle, brain, adipose tissue, heart and pancreas cells, and this receptor has been considered an important drug target in the treatment of diabetes. Administration of recently identified small-molecule GCGR antagonists in patients with type 2 diabetes results in a substantial reduction of fasting and postprandial glucose concentrations. Although an X-ray structure of the transmembrane domain of the GCGR has previously been solved, the ligand (NNC0640) was not resolved. Here we report the 2.5 Å structure of human GCGR in complex with the antagonist MK-0893 (ref. 4), which is found to bind to an allosteric site outside the seven transmembrane (7TM) helical bundle in a position between TM6 and TM7 extending into the lipid bilayer. Mutagenesis of key residues identified in the X-ray structure confirms their role in the binding of MK-0893 to the receptor. The unexpected position of the binding site for MK-0893, which is structurally similar to other GCGR antagonists, suggests that glucagon activation of the receptor is prevented by restriction of the outward helical movement of TM6 required for G-protein coupling. Structural knowledge of class B receptors is limited, with only one other ligand-binding site defined--for the corticotropin-releasing hormone receptor 1 (CRF1R)--which was located deep within the 7TM bundle. We describe a completely novel allosteric binding site for class B receptors, providing an opportunity for structure-based drug design for this receptor class and furthering our understanding of the mechanisms of activation of these receptors.

Exogenous 5-aminolevulinic acid alleviates low-temperature injury by regulating glutathione metabolism and ß-alanine metabolism in tomato seedling roots.

Food availability represents a major worldwide concern due to climate change and population growth. Low-temperature stress (LTS) severely restricts the growth of tomato seedlings. Exogenous 5-aminolevulinic acid (ALA) can alleviate the harm of abiotic stress including LTS; however, data on its protective mechanism on tomato seedling roots, the effects of organelle structure, and the regulation of metabolic pathways under LTS are lacking. In this study, we hope to fill the above gaps by exploring the effects of exogenous ALA on morphology, mitochondrial ultrastructure, reactive oxygen species (ROS) enrichment, physiological indicators, related gene expression, and metabolic pathway in tomato seedlings root under LTS. Results showed that ALA pretreatment could increase the activity of antioxidant enzymes and the content of antioxidant substances in tomato seedlings roots under LTS to scavenge the massively accumulated ROS, thereby protecting the mitochondrial structure of roots and promoting root development under LTS. Combined transcriptomic and metabolomic analysis showed that exogenous ALA pretreatment activated the glutathione metabolism and ß-alanine metabolism of tomato seedling roots under LTS, further enhanced the scavenging ability of tomato seedling roots to ROS, and improved the low-temperature tolerance of tomato seedlings. The findings provide a new insight into the regulation of the low-temperature tolerance of tomato by exogenous ALA.