The serine phosphorylations in the IRS-1 PIR domain abrogate IRS-1 and IR interaction.

Serine phosphorylations on insulin receptor substrate 1 (IRS-1) by diverse kinases aoccur widely during obesity-, stress-, and inflammation-induced conditions in models of insulin resistance and type 2 diabetes. In this study, we define a region within the human IRS-1, which is directly C-terminal to the PTB domain encompassing numerous serine phosphorylation sites including Ser307 (mouse Ser302) and Ser312 (mouse 307) creating a phosphorylation insulin resistance (PIR) domain. We demonstrate that the IRS-1 PTB-PIR with its unphosphorylated serine residues interacts with the insulin receptor (IR) but loses the IR-binding when they are phosphorylated. Surface plasmon resonance studies further confirm that the PTB-PIR binds stronger to IR than just the PTB domain, and that phosphorylations at Ser307, Ser312, Ser315, and Ser323 within the PIR domain result in abrogating the binding. Insulin-responsive cells containing the mutant IRS-1 with all these four serines changed into glutamates to mimic phosphorylations show decreased levels of phosphorylations in IR, IRS-1, and AKT compared to the wild-type IRS-1. Hydrogen-deuterium exchange mass spectrometry experiments indicating the PIR domain interacting with the N-terminal lobe and the hinge regions of the IR kinase domain further suggest the possibility that the IRS-1 PIR domain protects the IR from the PTP1B-mediated dephosphorylation.

RIPK3 and RIPK1 gene expression in pterygium: unveiling molecular insights into pathogenesis.

BACKGROUND: Pterygium, characterized by the abnormal proliferation of epithelial cells, matrix remodeling, vascularization, and lesion migration, is a prevalent ocular surface disease involving the growth of fibrovascular tissue on the cornea. Despite the unclear underlying causes of pterygium, numerous investigations have indicated the involvement of cell death pathways in the regulation of cell cycle dynamics. Consequently, the objective of this study was to assess the expression levels of necroptosis markers in individuals diagnosed with pterygium, aiming to shed light on the potential role of necroptosis in the pathogenesis of this condition. METHODS: This study aimed to investigate the expression patterns of receptor-interacting serine/threonine kinase 3 (RIPK3) and receptor-interacting serine/threonine kinase 1 (RIPK1) genes in pterygium tissues. 41 patients undergoing pterygium excision surgery were recruited. Resected pterygium samples and normal conjunctival tissues were collected, and RIPK3 and RIPK1 mRNA levels were measured using quantitative real-time PCR. RESULTS: Our findings reveal that the expression of RIPK3 is significantly increased in samples obtained from individuals with pterygium. However, no significant alterations were observed in the expression of RIPK1 in these samples. Results showed significantly higher RIPK3 expression in pterygium tissues compared to controls. Moreover, increased RIPK3 levels correlated negatively with pterygium recurrence rates. CONCLUSIONS: These findings suggest RIPK3 may play a protective role against pterygium recurrence through necroptosis.

Functions and Metabolism of Amino Acids in the Hair and Skin of Dogs and Cats.

The hair and skin of domestic cats or dogs account for 2% and 12-24% of their body weight, respectively, depending on breed and age. These connective tissues contain protein as the major constituent and provide the first line of defense against external pathogens and toxins. Maintenance of the skin and hair in smooth and elastic states requires special nutritional support, particularly an adequate provision of amino acids (AAs). Keratin (rich in cysteine, serine and glycine) is the major protein both in the epidermis of the skin and in the hair. Filaggrin [rich in some AAs (e.g., serine, glutamate, glutamine, glycine, arginine, and histidine)] is another physiologically important protein in the epidermis of the skin. Collagen and elastin (rich in glycine and proline plus 4-hydroxyproline) are the predominant proteins in the dermis and hypodermis of the skin. Taurine and 4-hydroxyproline are abundant free AAs in the skin of dogs and cats, and 4-hydroxyproline is also an abundant free AA in their hair. The epidermis of the skin synthesizes melanin (the pigment in the skin and hair) from tyrosine and produces trans-urocanate from histidine. Qualitative requirements for proteinogenic AAs are similar between cats and dogs but not identical. Both animal species require the same AAs to nourish the hair and skin but the amounts differ. Other factors (e.g., breeds, coat color, and age) may affect the requirements of cats or dogs for nutrients. The development of a healthy coat, especially a black coat, as well as healthy skin critically depends on AAs [particularly arginine, glycine, histidine, proline, 4-hydroxyproline, and serine, sulfur AAs (methionine, cysteine, and taurine), phenylalanine, and tyrosine] and creatine. Although there are a myriad of studies on AA nutrition in cats and dogs, there is still much to learn about how each AA affects the growth, development and maintenance of the hair and skin. Animal-sourced foodstuffs (e.g., feather meal and poultry by-product meal) are excellent sources of the AAs that are crucial to maintain the normal structure and health of the skin and hair in dogs and cats.

Roles of Nutrients in the Brain Development, Cognitive Function, and Mood of Dogs and Cats.

The brain is the central commander of all physical activities and the expression of emotions in animals. Its development and cognitive health critically depend on the neural network that consists of neurons, glial cells (namely, non-neuronal cells), and neurotransmitters (communicators between neurons). The latter include proteinogenic amino acids (e.g., L-glutamate, L-aspartate, and glycine) and their metabolites [e.g., γ-aminobutyrate, D-aspartate, D-serine, nitric oxide, carbon monoxide, hydrogen sulfide, and monoamines (e.g., dopamine, norepinephrine, epinephrine, and serotonin)]. In addition, some non-neurotransmitter metabolites of amino acids, such as taurine, creatine, and carnosine, also play important roles in brain development, cognitive health, behavior, and mood of dogs and cats. Much evidence shows that cats require dietary ω3 (α-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid) and ω6 (linoleic acid and arachidonic acid) polyunsaturated fatty acids for the development of the central nervous system. As an essential component of membranes of neurons and glial cells, cholesterol is also crucial for cognitive development and function. In addition, vitamins and minerals are required for the metabolism of AAs, lipids, and glucose in the nervous system, and also act as antioxidants. Thus, inadequate nutrition will lead to mood disorders. Some amino acids (e.g., arginine, glycine, methionine, serine, taurine, tryptophan, and tyrosine) can help to alleviate behavioral and mood disorders (e.g., depression, anxiety and aggression). As abundant providers of all these functional amino acids and lipids, animal-sourced foods (e.g., liver, intestinal mucosa, and meat) play important roles in brain development, cognitive function, and mood of dogs and cats. This may explain, in part, why dogs and cats prefer to eat visceral organs of their prey. Adequate provision of nutrients in all phases of the life cycle (pregnancy, lactation, postnatal growth, and adulthood) is essential for optimizing neurological health, while preventing cognitive dysfunction and abnormal behavior.

Numerous Serine/Threonine Kinases Affect Blood Cell Homeostasis in Drosophila melanogaster.

Blood cells in Drosophila serve primarily innate immune responses. Various stressors influence blood cell homeostasis regarding both numbers and the proportion of blood cell types. The principle molecular mechanisms governing hematopoiesis are conserved amongst species and involve major signaling pathways like Notch, Toll, JNK, JAK/Stat or RTK. Albeit signaling pathways generally rely on the activity of protein kinases, their specific contribution to hematopoiesis remains understudied. Here, we assess the role of Serine/Threonine kinases with the potential to phosphorylate the transcription factor Su(H) in crystal cell homeostasis. Su(H) is central to Notch signal transduction, and its inhibition by phosphorylation impedes crystal cell formation. Overall, nearly twenty percent of all Drosophila Serine/Threonine kinases were studied in two assays, global and hemocyte-specific overexpression and downregulation, respectively. Unexpectedly, the majority of kinases influenced crystal cell numbers, albeit only a few were related to hematopoiesis so far. Four kinases appeared essential for crystal cell formation, whereas most kinases restrained crystal cell development. This group comprises all kinase classes, indicative of the complex regulatory network underlying blood cell homeostasis. The rather indiscriminative response we observed opens the possibility that blood cells measure their overall phospho-status as a proxy for stress-signals, and activate an adaptive immune response accordingly.

Phosphorylation of Mad1 at serine 18 by Mps1 is required for the full virulence of rice blast fungus, Magnaporthe oryzae.

The spindle assembly checkpoint (SAC) proteins are conserved among eukaryotes safeguarding chromosome segregation fidelity during mitosis. However, their biological functions in plant-pathogenic fungi remain largely unknown. In this study, we found that the SAC protein MoMad1 in rice blast fungus (Magnaporthe oryzae) localizes on the nuclear envelope and is dispensable for M. oryzae vegetative growth and tolerance to microtubule depolymerizing agent treatment. MoMad1 plays an important role in M. oryzae infection-related development and pathogenicity. The monopolar spindle 1 homologue in M. oryzae (MoMps1) interacts with MoMad1 through its N-terminal domain and phosphorylates MoMad1 at Ser-18, which is conserved within the extended N termini of Mad1s from fungal plant pathogens. This phosphorylation is required for maintaining MoMad1 protein abundance and M. oryzae full virulence. Similar to the deletion of MoMad1, treatment with Mps1-IN-1 (an Mps1 inhibitor) caused compromised appressorium formation and decreased M. oryzae virulence, and these defects were dependent on its attenuating MoMad1 Ser-18 phosphorylation. Therefore, our study indicates the function of Mad1 in rice blast fungal pathogenicity and sheds light on the potential of blocking Mad1 phosphorylation by Mps1 to control crop fungal diseases.

Experimental evidence of d-glutamate racemase activity in the uncultivated bacterium Candidatus Saccharimonas aalborgensis.

The Candidate Phyla Radiation (CPR) encompasses widespread uncultivated bacteria with reduced genomes and limited metabolic capacities. Most CPR bacteria lack the minimal set of enzymes required for peptidoglycan (PG) synthesis, leaving it unclear how these bacteria produce this essential envelope component. In this study, we analysed the distribution of d-amino acid racemases that produce the universal PG components d-glutamate (d-Glu) or d-alanine (d-Ala). We also examined moonlighting enzymes that synthesize d-Glu or d-Ala. Unlike other phyla in the domain Bacteria, CPR bacteria do not exhibit these moonlighting activities and have, at most, one gene encoding either a Glu or Ala racemase. One of these 'orphan' racemases is a predicted Glu racemase (MurICPR) from the CPR bacterium Candidatus Saccharimonas aalborgenesis. The expression of MurICPR restores the growth of a Salmonella d-Glu auxotroph lacking its endogenous racemase and results in the substitution of l-Ala by serine as the first residue in a fraction of the PG stem peptides. In vitro, MurICPR exclusively racemizes Glu as a substrate. Therefore, Ca. Saccharimonas aalborgensis may couple Glu racemization to serine and d-Glu incorporation into the stem peptide. Our findings provide the first insights into the synthesis of PG by an uncultivated environmental bacterium and illustrate how to experimentally test enzymatic activities from CPR bacteria related to PG metabolism.

A molecular dynamics simulation study of glycine/serine octapeptides labeled with 2,3-diazabicyclo[2.2.2]oct-2-ene fluorophore.

The compound 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) is a versatile fluorophore widely used in Förster resonance energy transfer (FRET) spectroscopy studies due to its remarkable sensitivity, enabling precise donor-acceptor distance measurements, even for short peptides. Integrating time-resolved and FRET spectroscopies with molecular dynamics simulations provides a robust approach to unravel the structure and dynamics of biopolymers in a solution. This study investigates the structural behavior of three octapeptide variants: Trp-(Gly-Ser)3-Dbo, Trp-(GlyGly)3-Dbo, and Trp-(SerSer)3-Dbo, where Dbo represents the DBO-containing modified aspartic acid, using molecular dynamics simulations. Glycine- and serine-rich amino acid fragments, common in flexible protein regions, play essential roles in functional properties. Results show excellent agreement between end-to-end distances, orientational factors from simulations, and the available experimental and theoretical data, validating the reliability of the GROMOS force field model. The end-to-end distribution, modeled using three Gaussian distributions, reveals a complex shape, confirmed by cluster analysis highlighting a limited number of significant conformations dominating the peptide landscape. All peptides predominantly adopt a disordered state in the solvent, yet exhibit a compact shape, aligning with the model of disordered polypeptide chains in poor solvents. Conformations show marginal dependence on chain composition, with Ser-only chains exhibiting slightly more elongation. This study enhances our understanding of peptide behavior, providing valuable insights into their structural dynamics in solution.

Cryo-EM structure of the human Asc-1 transporter complex.

The Alanine-Serine-Cysteine transporter 1 (Asc-1 or SLC7A10) forms a crucial heterodimeric transporter complex with 4F2hc (SLC3A2) through a covalent disulfide bridge. This complex enables the sodium-independent transport of small neutral amino acids, including L-Alanine (L-Ala), Glycine (Gly), and D-Serine (D-Ser), within the central nervous system (CNS). D-Ser and Gly are two key endogenous glutamate co-agonists that activate N-methyl-d-aspartate (NMDA) receptors by binding to the allosteric site. Mice deficient in Asc-1 display severe symptoms such as tremors, ataxia, and seizures, leading to early postnatal death. Despite its physiological importance, the functional mechanism of the Asc-1-4F2hc complex has remained elusive. Here, we present cryo-electron microscopy (cryo-EM) structures of the human Asc-1-4F2hc complex in its apo state, D-Ser bound state, and L-Ala bound state, resolved at 3.6 Å, 3.5 Å, and 3.4 Å, respectively. Through detailed structural analysis and transport assays, we uncover a comprehensive alternating access mechanism that underlies conformational changes in the complex. In summary, our findings reveal the architecture of the Asc-1 and 4F2hc complex and provide valuable insights into substrate recognition and the functional cycle of this essential transporter complex.

Disentangling the formation, mechanism, and evolvement of the covalent methanesulfonyl fluoride acetylcholinesterase adduct: Insights into an aged-like inactive complex susceptible to reactivation by a combination of nucleophiles.

Chemical warfare nerve agents and pesticides, known as organophosphorus compounds inactivate cholinesterases (ChEs) by phosphorylating the serine hydroxyl group located at the active site of ChEs. Over the course of time, phosphorylation is followed by loss of an organophosphate-leaving group and the bond with ChEs becomes irreversible, a process known as aging. Differently, structurally related irreversible catalytic poisons bearing sulfur instead of phosphorus convert ChEs in its aged form only by covalently binding to the key catalytic serine. Kinetic and crystallographic studies of the interaction between Torpedo californica acetylcholinesterase (TcAChE) and a small organosulfonate, methanesulfonyl fluoride (MSF), indeed revealed irreversibly methylsulfonylated serine 200, to be isosteric with the bound aged sarin/soman analogues. The potent bulky reversible inhibitor 7-bis-tacrine (BTA) adopts, in the active site of the crystal structure of the MSF-enzyme adduct, a location and an orientation that closely resemble the one being found in the crystal structure of the BTA-enzyme complex. Remarkably, the presence of BTA accelerates the rate of methanesulfonylation by a factor of two. This unexpected result can be explained on the basis of two facts: i) the steric hindrance exerted by BTA to MSF in accessing the active site and ii) the acceleration of the MSF-enzyme adduct formation as a consequence of the lowering of the rotational and translational degrees of freedom in the proximity of the catalytic serine. It is well known that pralidoxime (2-Pyridine Aldoxime Methyl chloride, 2-PAM) alone or in the presence of the substrate acetylcholine cannot reactivate the active site serine of the TcAChE-MSF adduct. We show that the simultaneous presence of 2-PAM and the additional neutral oxime, 2-[(hydroxyimino)methyl]-l-methylimidazol (2-HAM), triggers the reactivation process of TcAChE within the hour timescale. Overall, our results pave the way toward the likely use of a cocktail of distinctive oximes as a promising recipe for an effective and fast reactivation of aged cholinesterases.

A study on the significance of serine hydroxymethyl transferase expression and its role in bladder cancer.

Bladder cancer (BLCA) is a common malignant tumor in urinary system all over the world. However, due to its high recurrence rate and complex causes, clinicians often have limited options for surgical and drug treatments. Recent researchs on the molecular mechanism of BLCA have reveals its biological progress and potential for early diagnosis. Serine hydroxymethyltransferase 1/2 (SHMT1/2) is a crucial enzyme in the one-carbon metabolism of tumor cells, and the expression levels of these isozymes have been found to be associated with the biological progression of various malignant tumors. However, the impact of SHMT1/2 on the biological progression of bladder cancer and its molecular regulation mechanism remain unclear. In this research utilizes BLCA clinical sample data, the TCGA database, and in vitro cell experiments to predict the expression levels of SHMT1/2 in BLCA. The findings indicate that SHMT1 remained unchanged, while SHMT2 expression is increased in BLCA, which was related to poor prognosis. Additionally, SHMT2 affects the growth, migration, and apoptosis of bladder cancer cells in vitro. It also influences the expression levels of E-cadherin and N-cadherin, ultimately impacting the malignant biological progression of bladder tumors. These results establish a correlation between SHMT2 and the malignant biological progression of BLCA, providing a theoretical basis for the early diagnosis and treatment of bladder cancer.

Identification of benzo[b]thiophene-1,1-dioxide derivatives as novel PHGDH covalent inhibitors.

The increased de novo serine biosynthesis confers many advantages for tumorigenesis and metastasis. Phosphoglycerate dehydrogenase (PHGDH), a rate-limiting enzyme in serine biogenesis, exhibits hyperactivity across multiple tumors and emerges as a promising target for cancer treatment. Through screening our in-house compound library, we identified compound Stattic as a potent PHGDH inhibitor (IC50 = 1.98 ± 0.66 µM). Subsequent exploration in structural activity relationships led to the discovery of compound B12 that demonstrated the increased enzymatic inhibitory activity (IC50 = 0.29 ± 0.02 µM). Furthermore, B12 exhibited robust inhibitory effects on the proliferation of MDA-MB-468, NCI-H1975, HT1080 and PC9 cells that overexpress PHGDH. Additionally, using a [U-13C6]-glucose tracing assay, B12 was found to reduce the production of glucose-derived serine in MDA-MB-468 cells. Finally, mass spectrometry-based peptide profiling, mutagenesis experiment and molecular docking study collectively suggested that B12 formed a covalent bond with Cys421 of PHGDH.

Combinatorially restricted computational design of protein-protein interfaces to produce IgG heterodimers.

Redesigning protein-protein interfaces is an important tool for developing therapeutic strategies. Interfaces can be redesigned by in silico screening, which allows for efficient sampling of a large protein space before experimental validation. However, computational costs limit the number of combinations that can be reasonably sampled. Here, we present combinatorial tyrosine (Y)/serine (S) selection (combYSelect), a computational approach combining in silico determination of the change in binding free energy (ΔΔG) of an interface with a highly restricted library composed of just two amino acids, tyrosine and serine. We used combYSelect to design two immunoglobulin G (IgG) heterodimers-combYSelect1 (L368S/D399Y-K409S/T411Y) and combYSelect2 (D399Y/K447S-K409S/T411Y)-that exhibit near-optimal heterodimerization, without affecting IgG stability or function. We solved the crystal structures of these heterodimers and found that dynamic π-stacking interactions and polar contacts drive preferential heterodimeric interactions. Finally, we demonstrated the utility of our combYSelect heterodimers by engineering both a bispecific antibody and a cytokine trap for two unique therapeutic applications.

STK16 promoted colorectal cancer progress in a c-MYC signaling-dependent manner.

BACKGROUND: Colorectal cancer standed as a global health challenge, ranking third in cancer incidence and second in cancer-related deaths worldwide. A deeper understanding of the intricate mechanisms driving colorectal cancer development was pressing need. STK16 had garnered attention in recent researches, while its involvement in cancer had been minimally explored. c-MYC had emerged as a key player in cancer biology. Due to its complex structure, multifunctionality, and intricate interactions, directly inhibiting the activity of c-MYC proves to be challenging. Hence, current research was directing efforts towards modulating c-MYC expression levels. METHODS: Immunoblot, Immunohistochemistry and immunoprecipitation assays were conducted to assess the indicated protein expression levels. RT-PCR was performed to detect the corresponding mRNA expression levels. The proliferation, migration, invasion, and colony formation abilities of the specified cancer cells were investigated using CCK8 assays, Brdu assays, transwell assays, and colony formation assays, respectively. Cellular and animal experiments were performed to investigate the correlation between STK16 signaling and c-MYC signaling. RESULTS: STK16 plays a positive regulatory role in the progression of colorectal cancer. Delving into the molecular mechanisms, we unveiled that STK16 phosphorylated c-MYC at serine 452, a pivotal event hindering the ubiquitin-proteasome pathway degradation of c-MYC. Importantly, colorectal cancer proliferation mediated by STK16 was found to be dependent on the phosphorylation of c-MYC at S452. Furthermore, the researchers demonstrated that STK16 knockout or pharmacological inhibition significantly curtailed colorectal cancer proliferation and c-MYC expression in in vivo animal models. CONCLUSION: We discovered that STK16 phosphorylates c-MYC at serine 452, hindering its degradation via the ubiquitin-proteasome pathway. STK16 inhibition, either genetically or pharmacologically, effectively curtails cancer growth and c-MYC expression in vivo. These findings highlight STK16 as a potential therapeutic target for colorectal cancer.

Tetrahydrofolate levels influence 2-aminoacrylate stress in Salmonella enterica.

In Salmonella enterica, the absence of the RidA deaminase results in the accumulation of the reactive enamine 2-aminoacrylate (2AA). The resulting 2AA stress impacts metabolism and prevents growth in some conditions by inactivating a specific target pyridoxal 5'-phosphate (PLP)-dependent enzyme(s). The detrimental effects of 2AA stress can be overcome by changing the sensitivity of a critical target enzyme or modifying flux in one or more nodes in the metabolic network. The catabolic L-alanine racemase DadX is a target of 2AA, which explains the inability of an alr ridA strain to use L-alanine as the sole nitrogen source. Spontaneous mutations that suppressed the growth defect of the alr ridA strain were identified as lesions in folE, which encodes GTP cyclohydrolase and catalyzes the first step of tetrahydrofolate (THF) synthesis. The data here show that THF limitation resulting from a folE lesion, or inhibition of dihydrofolate reductase (FolA) by trimethoprim, decreases the 2AA generated from endogenous serine. The data are consistent with an increased level of threonine, resulting from low folate levels, decreasing 2AA stress.IMPORTANCERidA is an enamine deaminase that has been characterized as preventing the 2-aminoacrylate (2AA) stress. In the absence of RidA, 2AA accumulates and damages various cellular enzymes. Much of the work describing the 2AA stress system has depended on the exogenous addition of serine to increase the production of the enamine stressor. The work herein focuses on understanding the effect of 2AA stress generated from endogenous serine pools. As such, this work describes the consequences of a subtle level of stress that nonetheless compromises growth in at least two conditions. Describing mechanisms that alter the physiological consequences of 2AA stress increases our understanding of endogenous metabolic stress and how the robustness of the metabolic network allows perturbations to be modulated.

Standardized ileal digestibility of amino acids in Chinese fermented soybean meal from different sources fed to mid and late-gestating sows.

We determined apparent ileal digestibility (AID) and standardized ileal digestibility (SID) values of crude protein (CP) and amino acids (AA) in fermented soybean meal from five different sources (FSBM 1 to 5) in China when fed to mid and late-gestating sows. Twenty-four parity four sows (12 at 30 d in gestation and 12 at 80 d in gestation) were fitted with a T-cannula in the distal ileum and used in this experiment. Sows were randomly assigned to a replicated 6 × 3 Youden square design including six diets and three periods. Six diets were provided for sows in mid and late gestation, including a nitrogen-free diet and five test diets containing 26% FSBM from different sources. Results showed that there were differences in AID and SID of CP among the different FSBM samples, but no differences between sow physiological stages were observed. Specifically, when mid-gestating sows were fed FSBM 2, the AID of CP was the lowest, whereas FSBM 3 exhibited a greater AID of CP when compared to the other FSBM samples (P < 0.01). Furthermore, during late gestation, FSBM 3 consistently had greater SID of CP when compared to other FSBM samples (P < 0.01). The ileal digestibility of most AA varied with different FSBM samples. In both mid and late gestation, differences (P < 0.05) were observed for AID of lysine, tryptophan, histidine, and arginine across different FSBM samples. Similarly, the AID of dispensable AA (cysteine, glutamine, and serine) also exhibited differences (P < 0.05) across different FSBM samples in both mid and late-gestating sows. For mid-gestating sows, SID differences relating to lysine, phenylalanine, tryptophan, threonine, and arginine were observed among different diets (P < 0.05). In late-gestating sows, SID values for lysine, tryptophan, leucine, and arginine differed across diets (P < 0.05). Furthermore, the ileal digestibility of some dispensable AA was influenced by physiological stage, as evidenced by greater AID and SID values for glycine, glutamine, cysteine, and serine in late-gestating sows when compared to mid-gestating sows (P < 0.01). In summary, our study determined AA ileal digestibility of different FSBM fed to mid and late-gestating sows. We observed that the AA ileal digestibility differed among five FSBM samples, but the physiological stage of sows did not affect the ileal digestibility of CP and most AA. Additionally, when formulating diets for sows, it is crucial to consider the nutritional value differences of FSBM.

Fermented soybean meal (FSBM) is obtained from the microbial fermentation of soybean meal, which reduces anti-nutritional factor levels and enhances other nutrient content. Substituting soybean meal with FSBM in piglet and growing pig diets improves nutrient digestibility. However, its nutritional value for sows remains unclear. Therefore, five sources of FSBM were fed to sows in mid and late gestation to evaluate apparent ileal digestibility (AID) and standardized ileal digestibility (SID) values of amino acids (AA). We found that different FSBM samples impacted the SID value of AA when fed to gestating sows. Additionally, sow physiological stage influenced the SID of some dispensable AA. These findings provide valuable insights into the incorporation of FSBM into sow diets.

Serine enrichment in tumors promotes regulatory T cell accumulation through sphinganine-mediated regulation of c-Fos.

CD4+ regulatory T (Treg) cells accumulate in the tumor microenvironment (TME) and suppress the immune system. Whether and how metabolite availability in the TME influences Treg cell differentiation is not understood. Here, we measured 630 metabolites in the TME and found that serine and palmitic acid, substrates required for the synthesis of sphingolipids, were enriched. A serine-free diet or a deficiency in Sptlc2, the rate-limiting enzyme catalyzing sphingolipid synthesis, suppressed Treg cell accumulation and inhibited tumor growth. Sphinganine, an intermediate metabolite in sphingolipid synthesis, physically interacted with the transcription factor c-Fos. Sphinganine c-Fos interactions enhanced the genome-wide recruitment of c-Fos to regions near the transcription start sites of target genes including Pdcd1 (encoding PD-1), which promoted Pdcd1 transcription and increased inducible Treg cell differentiation in vitro in a PD-1-dependent manner. Thus, Sptlc2-mediated sphingolipid synthesis translates the extracellular information of metabolite availability into nuclear signals for Treg cell differentiation and limits antitumor immunity.

Treatment of Organophosphorus Poisoning with 6-Alkoxypyridin-3-ol Quinone Methide Precursors: Resurrection of Methylphosphonate-Aged Acetylcholinesterase.

Organophosphorus (OP) nerve agents inhibit acetylcholinesterase (AChE), creating a cholinergic crisis in which death can occur. The phosphylated serine residue spontaneously dealkylates to the OP-aged form, which current therapeutics cannot reverse. Soman's aging half-life is 4.2 min, so immediate recovery (resurrection) of OP-aged AChE is needed. In 2018, we showed pyridin-3-ol-based quinone methide precursors (QMPs) can resurrect OP-aged electric eel AChE in vitro, achieving 2% resurrection after 24 h of incubation (pH 7, 4 mM). We prepared 50 unique 6-alkoxypyridin-3-ol QMPs with 10 alkoxy groups and five amine leaving groups to improve AChE resurrection. These compounds are predicted in silico to cross the blood-brain barrier and treat AChE in the central nervous system. This library resurrected 7.9% activity of OP-aged recombinant human AChE after 24 h at 250 µM, a 4-fold increase from our 2018 report. The best QMP (1b), with a 6-methoxypyridin-3-ol core and a diethylamine leaving group, recovered 20.8% (1 mM), 34% (4 mM), and 42.5% (predicted maximum) of methylphosphonate-aged AChE activity over 24 h. Seven QMPs recovered activity from AChE aged with Soman and a VX degradation product (EA-2192). We hypothesize that QMPs form the quinone methide (QM) to realkylate the phosphylated serine residue as the first step of resurrection. We calculated thermodynamic energetics for QM formation, but there was no trend with the experimental biochemical data. Molecular docking studies revealed that QMP binding to OP-aged AChE is not the determining factor for the observed biochemical trends; thus, QM formation may be enzyme-mediated.

Mendelian Randomization Study With Clinical Follow-Up Links Metabolites to Risk and Severity of Pulmonary Arterial Hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) exhibits phenotypic heterogeneity and variable response to therapy. The metabolome has been implicated in the pathogenesis of PAH, but previous works have lacked power to implicate specific metabolites. Mendelian randomization (MR) is a method for causal inference between exposures and outcomes. METHODS AND RESULTS: Using genome-wide association study summary statistics, we implemented MR analysis to test for potential causal relationships between serum concentration of 575 metabolites and PAH. Five metabolites were causally associated with the risk of PAH after multiple testing correction. Next, we measured serum concentration of candidate metabolites in an independent clinical cohort of 449 patients with PAH to check whether metabolite concentrations are correlated with markers of disease severity. Of the 5 candidates nominated by our MR work, serine was negatively associated and homostachydrine was positively associated with clinical severity of PAH via direct measurement in this independent clinical cohort. Finally we used conditional and orthogonal approaches to explore the biology underlying our lead metabolites. Rare variant burden testing was carried out using whole exome sequencing data from 578 PAH cases and 361 675 controls. Multivariable MR is an extension of MR that uses a single set of instrumental single-nucleotide polymorphisms to measure multiple exposures; multivariable MR is used to determine interdependence between the effects of different exposures on a single outcome. Rare variant analysis demonstrated that loss-of-function mutations within activating transcription factor 4, a transcription factor responsible for upregulation of serine synthesis under conditions of serine starvation, are associated with higher risk for PAH. Homostachydrine is a xenobiotic metabolite that is structurally related to l-proline betaine, which has previously been linked to modulation of inflammation and tissue remodeling in PAH. Our multivariable MR analysis suggests that the effect of l-proline betaine is actually mediated indirectly via homostachydrine. CONCLUSIONS: Our data present a method for study of the metabolome in the context of PAH, and suggests several candidates for further evaluation and translational research.

MbovP0725, a secreted serine/threonine phosphatase, inhibits the host inflammatory response and affects metabolism in Mycoplasma bovis.

Mycoplasma species are able to produce and release secreted proteins, such as toxins, adhesins, and virulence-related enzymes, involved in bacteria adhesion, invasion, and immune evasion between the pathogen and host. Here, we investigated a novel secreted protein, MbovP0725, from Mycoplasma bovis encoding a putative haloacid dehalogenase (HAD) hydrolase function of a key serine/threonine phosphatase depending on Mg2+ for the dephosphorylation of its substrate pNPP, and it was most active at pH 8 to 9 and temperatures around 40°C. A transposon insertion mutant strain of M. bovis HB0801 that lacked the protein MbovP0725 induced a stronger inflammatory response but with a partial reduction of adhesion ability. Using transcriptome sequencing and quantitative reverse transcription polymerase chain reaction analysis, we found that the mutant was upregulated by the mRNA expression of genes from the glycolysis pathway, while downregulated by the genes enriched in ABC transporters and acetate kinase-phosphate acetyltransferase pathway. Untargeted metabolomics showed that the disruption of the Mbov_0725 gene caused the accumulation of 9-hydroxyoctadecadienoic acids and the consumption of cytidine 5'-monophosphate, uridine monophosphate, and adenosine monophosphate. Both the exogenous and endogenous MbvoP0725 protein created by purification and transfection inhibited lipopolysaccharide (LPS)-induced IL-1ß, IL-6, and TNF-α mRNA production and could also attenuate the activation of MAPK-associated pathways after LPS treatment. A pull-down assay identified MAPK p38 and ERK as potential substrates for MbovP0725. These findings define metabolism- and virulence-related roles for a HAD family phosphatase and reveal its ability to inhibit the host pro-inflammatory response. IMPORTANCE: Mycoplasma bovis (M. bovis) infection is characterized by chronic pneumonia, otitis, arthritis, and mastitis, among others, and tends to involve the suppression of the immune response via multiple strategies to avoid host cell immune clearance. This study found that MbovP0725, a haloacid dehalogenase (HAD) family phosphatase secreted by M. bovis, had the ability to inhibit the host pro-inflammatory response induced by lipopolysaccharide. Transcriptomic and metabolomic analyses were used to identify MbovP0725 as an important phosphatase involved in glycolysis and nucleotide metabolism. The M. bovis transposon mutant strain T8.66 lacking MbovP0725 induced a higher inflammatory response and exhibited weaker adhesion to host cells. Additionally, T8.66 attenuated the phosphorylation of MAPK P38 and ERK and interacted with the two targets. These results suggested that MbovP0725 had the virulence- and metabolism-related role of a HAD family phosphatase, performing an anti-inflammatory response during M. bovis infection.