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ACS Synthetic Biology Jun 2024Glycosylation is a ubiquitous modification present across all of biology, affecting many things such as physicochemical properties, cellular recognition, subcellular... (Review)
Review
Glycosylation is a ubiquitous modification present across all of biology, affecting many things such as physicochemical properties, cellular recognition, subcellular localization, and immunogenicity. Nucleotide sugars are important precursors needed to study glycosylation and produce glycosylated products. is a potentially powerful platform for producing glycosylated biomolecules, but it lacks nucleotide sugar diversity. Nucleotide sugar metabolism is complex, and understanding how to engineer it will be necessary to both access and study heterologous glycosylations found across biology. This review overviews the potential challenges with engineering nucleotide sugar metabolism in yeast from the salvage pathways that convert free sugars to their associated UDP-sugars to synthesis where nucleotide sugars are interconverted through a complex metabolic network with governing feedback mechanisms. Finally, recent examples of engineering complex glycosylation of small molecules in are explored and assessed.
Topics: Saccharomyces cerevisiae; Glycosylation; Metabolic Engineering; Biological Products; Nucleotides; Metabolic Networks and Pathways
PubMed: 38820348
DOI: 10.1021/acssynbio.3c00737 -
International Journal of Medical... 2024Citicoline can be used to reduce acute ischemic stroke injury via venous infusion, however, its protective effects in the brain extracellular space remain largely...
Citicoline can be used to reduce acute ischemic stroke injury via venous infusion, however, its protective effects in the brain extracellular space remain largely unknown. Herein, we investigated the brain protective effects of citicoline administered via the brain extracellular space and sought precise effective dosage range that can protect against ischemic injury after experimental ischemic stroke in rats. : Fifty-six Sprague-Dawley rats were randomly divided into control, intraperitoneal (IP), caudate-putamen (CPu)-25, CPu-40, CPu-50, CPu-60 and CPu-75 groups based on the infusion site and concentration of citicoline. Two hours after the administration of citicoline, the rats were subjected to a permanent middle cerebral artery occlusion to mimic acute ischemic stroke. Then, the brain infarct volume in rats after stroke was measured and their neurological deficiency was evaluated to explain the protective effects and effective dosage range of citicoline. Compared to the control and IP groups, brain infarct volume of rats in CPu-40, CPu-50, and CPu-60 groups is significant smaller. Furthermore, the brain infarct volume of rats in CPu-50 is the least. Here, we showed that citicoline can decrease the brain infarct volume, thus protecting the brain from acute ischemic stroke injury. We also found that the appropriate effective citicoline dose delivered via the brain extracellular space is 50 mM. Our study provides novel insights into the precise treatment of acute ischemic stroke by citicoline via the brain extracellular space, further guiding the treatment of brain disease.
Topics: Animals; Cytidine Diphosphate Choline; Rats; Ischemic Stroke; Disease Models, Animal; Extracellular Space; Male; Rats, Sprague-Dawley; Brain; Neuroprotective Agents; Humans; Infarction, Middle Cerebral Artery; Brain Ischemia
PubMed: 38818467
DOI: 10.7150/ijms.93482 -
MLife Dec 2023Photosynthetic microalgae like hold enormous potential as sustainable, light-driven biofactories for the production of high-value natural products such as terpenoids....
Photosynthetic microalgae like hold enormous potential as sustainable, light-driven biofactories for the production of high-value natural products such as terpenoids. is distinguished as a particularly robust host with extensive genomic and transgenic resources available. Its capacity to grow in wastewater, brackish, and sea waters, coupled with advances in microalgal metabolic engineering, genome editing, and synthetic biology, provides an excellent opportunity. In the present work, we demonstrate how can be engineered to produce the diterpene casbene-an important intermediate in the biosynthesis of pharmacologically relevant macrocyclic diterpenoids. Casbene accumulated after stably expressing and targeting the casbene synthase from (DgTPS1) to the algal chloroplast. The engineered strains yielded production titers of up to 0.12 mg g total dry cell weight (DCW) casbene. Heterologous overexpression and chloroplast targeting of two upstream rate-limiting enzymes in the 2-C-methyl- d-erythritol 4-phosphate pathway, 1-deoxy- d-xylulose-5-phosphate synthase and geranylgeranyl diphosphate synthase genes, further enhanced the yield of casbene to a titer up to 1.80 mg g DCW. The results presented here form a basis for further development and production of complex plant diterpenoids in microalgae.
PubMed: 38818264
DOI: 10.1002/mlf2.12097 -
Research and Practice in Thrombosis and... Mar 2024Assessment of platelet function is key in diagnosing bleeding disorders and evaluating antiplatelet drug efficacy. However, there is a prevailing "one-size-fits-all"...
BACKGROUND
Assessment of platelet function is key in diagnosing bleeding disorders and evaluating antiplatelet drug efficacy. However, there is a prevailing "one-size-fits-all" approach in the interpretation of measures of platelet reactivity, with arbitrary cutoffs often derived from healthy volunteer responses.
OBJECTIVES
Our aim was to compare well-used platelet reactivity assays.
METHODS
Blood and platelet-rich plasma obtained from the Framingham Heart Study ( = 3429) were assayed using a range of agonists in 5 platelet assays: light transmission aggregometry, Optimul aggregometry, Multiplate impedance aggregometry (Roche Diagnostics), Total Thrombus-Formation Analysis System, and flow cytometry. Using linear mixed-effect models, we determined the contribution of preanalytical and technical factors that modulated platelet reactivity traits.
RESULTS
A strong intra-assay correlation of platelet traits was seen in all assays, particularly Multiplate velocity ( = 0.740; ristocetin vs arachidonic acid). In contrast, only moderate interassay correlations were observed ( = 0.375; adenosine diphosphate Optimul E vs light transmission aggregometry large area under the curve). As expected, antiplatelet drugs strongly reduced platelet responses, with aspirin use primarily targeting arachidonic acid-induced aggregation, and explained substantial variance (β = -1.735; = 4.59 × 10; variance proportion = 46.2%) and P2Y antagonists blocking adenosine diphosphate responses (β = -1.612; = 6.75 × 10; variance proportion = 2.1%). Notably, female sex and older age were associated with enhanced platelet reactivity. Fasting status and deviations from standard venipuncture practices did not alter platelet reactivity significantly. Finally, the agonist batch, phlebotomist, and assay technician (more so for assays that require additional sample manipulation) had a moderate to large effect on measured platelet reactivity.
CONCLUSION
Caution must be exercised when extrapolating findings between assays, and the use of standard ranges must be medication-specific and sex-specific at a minimum. Researchers should also consider preanalytical and technical variables when designing experiments and interpreting platelet reactivity measures.
PubMed: 38813256
DOI: 10.1016/j.rpth.2024.102406 -
Indian Journal of Pharmacology Mar 2024Sildenafil, a common over-the-counter pill often self-administered at high doses for erectile dysfunction, has been reported to rarely cause prothrombotic events and...
Off-target effect of high-dose sildenafil on adenosine 5'- diphosphate and collagen-induced platelet activation through mitogen-activated protein kinase pathway in treated BALB/C mice and in vitro experiments: A preliminary study.
Sildenafil, a common over-the-counter pill often self-administered at high doses for erectile dysfunction, has been reported to rarely cause prothrombotic events and sudden cardiac death in a few case reports. Therefore, we investigated the in vitro and in vivo effect of sildenafil treatment and dosage on platelet activation and mitogen-activated protein kinase (MAPK) phosphorylation. BALB/C mice were segregated into four groups, each having four mice each (control, low [3.25 mg/kg], medium [6.5 mg/kg], and high [13 mg/kg] sildenafil), and after the treatment, blood was drawn from each mouse and washed platelets prepared. Washed platelets were incubated with CD41 PE-Cy7 and Phospho-p38 MAPK PE antibodies and analyzed using a flow cytometer for platelet activation and adenosine 5'- diphosphate (ADP)/collagen-induced MAPK phosphorylation. Washed platelets obtained from the venous blood of 18 human volunteers, were incubated with PAC-1 FITC and Phospho-p38 MAPK PE antibodies, and platelet activation (ADP and collagen), followed by flow cytometry analysis. There was a significant increase in both platelet activation as well as MAPK phosphorylation in the presence of collagen in the high-dose (13 mg/kg) sildenafil group (P = 0.000774). Further, increased platelet activation was observed in samples that were treated with high-dose sildenafil as compared to the untreated samples (P < 0.00001). These studies show the risk of prothrombotic episodes in patients on high-dose sildenafil (100 mg), in those with even mild endothelial dysfunction due to ADP, and collagen-induced platelet activation through MAPK phosphorylation, which was not seen in the low-and intermediate-dose cohorts.
Topics: Animals; Sildenafil Citrate; Platelet Activation; Mice, Inbred BALB C; Male; Humans; Collagen; Mice; Adenosine Diphosphate; Blood Platelets; Phosphorylation; Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Phosphodiesterase 5 Inhibitors; Dose-Response Relationship, Drug; Adult
PubMed: 38808925
DOI: 10.4103/ijp.ijp_312_23 -
Journal of the American Heart... Jun 2024ADP and ATP are importantly involved in vascular and thrombotic homeostasis, via multiple receptor pathways. Blockade of ADP P2Y receptors inhibits platelet aggregation... (Randomized Controlled Trial)
Randomized Controlled Trial
Safety, Tolerability, and Pharmacodynamics of AZD3366 (Optimized Human CD39L3 Apyrase) Alone and in Combination With Ticagrelor and Acetylsalicylic Acid: A Phase 1, Randomized, Placebo-Controlled Study.
BACKGROUND
ADP and ATP are importantly involved in vascular and thrombotic homeostasis, via multiple receptor pathways. Blockade of ADP P2Y receptors inhibits platelet aggregation and represents an effective cardiovascular disease prevention strategy. AZD3366 (APT102), a long-acting recombinant form of an optimized CD39L3 human apyrase, has effectively reduced ATP, ADP, and platelet aggregation and provided tissue protection in preclinical models, features that could be very beneficial in treating patients with cardiovascular disease.
METHODS AND RESULTS
We conducted this phase 1, first-in-human study of single ascending doses of intravenous AZD3366 or placebo, including doses added to dual antiplatelet therapy with ticagrelor and acetylsalicylic acid. The primary objective was safety and tolerability; secondary and exploratory objectives included pharmacokinetics, pharmacodynamics (measured as inhibition of platelet aggregation), adenosine diphosphatase (ADPase) activity, and ATP/ADP metabolism. In total, 104 participants were randomized. AZD3366 was generally well tolerated, with no major safety concerns observed. ADPase activity increased in a dose-dependent manner with a strong correlation to AZD3366 exposure. Inhibition of ADP-stimulated platelet aggregation was immediate, substantial, and durable. In addition, there was a prompt decrease in systemic ATP concentration and an increase in adenosine monophosphate concentrations, whereas ADP concentration appeared generally unaltered. At higher doses, there was a prolongation of capillary bleeding time without detectable changes in the ex vivo thromboelastometric parameters.
CONCLUSIONS
AZD3366 was well tolerated in healthy participants and demonstrated substantial and durable inhibition of platelet aggregation after single dosing. Higher doses prolonged capillary bleeding time without detectable changes in ex vivo thromboelastometric parameters.
REGISTRATION
URL: https://www.clinicaltrials.gov; Unique Identifier: NCT04588727.
Topics: Humans; Male; Ticagrelor; Female; Apyrase; Platelet Aggregation; Aspirin; Platelet Aggregation Inhibitors; Middle Aged; Adult; Double-Blind Method; Dual Anti-Platelet Therapy; Drug Therapy, Combination; Young Adult; Adenosine Diphosphate; Blood Platelets; Dose-Response Relationship, Drug; Treatment Outcome; Recombinant Proteins; Purinergic P2Y Receptor Antagonists
PubMed: 38804212
DOI: 10.1161/JAHA.123.033985 -
Nature Communications May 2024Actin nucleotide-dependent actin remodeling is essential to orchestrate signal transduction and cell adaptation. Rapid energy starvation requires accurate and timely...
Actin nucleotide-dependent actin remodeling is essential to orchestrate signal transduction and cell adaptation. Rapid energy starvation requires accurate and timely reorganization of the actin network. Despite distinct treadmilling mechanisms of ADP- and ATP-actin filaments, their filament structures are nearly identical. How other actin-binding proteins regulate ADP-actin filament assembly is unclear. Here, we show that Spa2 which is the polarisome scaffold protein specifically remodels ADP-actin upon energy starvation in budding yeast. Spa2 triggers ADP-actin monomer nucleation rapidly through a dimeric core of Spa2 (aa 281-535). Concurrently, the intrinsically disordered region (IDR, aa 1-281) guides Spa2 undergoing phase separation and wetting on the surface of ADP-G-actin-derived F-actin and bundles the filaments. Both ADP-actin-specific nucleation and bundling activities of Spa2 are actin D-loop dependent. The IDR and nucleation core of Spa2 are evolutionarily conserved by coexistence in the fungus kingdom, suggesting a universal adaptation mechanism in the fungal kingdom in response to glucose starvation, regulating ADP-G-actin and ADP-F-actin with high nucleotide homogeneity.
Topics: Actin Cytoskeleton; Actins; Adenosine Diphosphate; Glucose; Microfilament Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 38802374
DOI: 10.1038/s41467-024-48863-4 -
Journal of the American Chemical Society Jun 2024Liquid-jet photoemission spectroscopy (LJ-PES) allows for a direct probing of electronic structure in aqueous solutions. We show the applicability of the approach to...
Liquid-jet photoemission spectroscopy (LJ-PES) allows for a direct probing of electronic structure in aqueous solutions. We show the applicability of the approach to biomolecules in a complex environment, exploring site-specific information on the interaction of adenosine triphosphate in the aqueous phase (ATP) with magnesium (Mg), highlighting the synergy brought about by the simultaneous analysis of different regions in the photoelectron spectrum. In particular, we demonstrate intermolecular Coulombic decay (ICD) spectroscopy as a new and powerful addition to the arsenal of techniques for biomolecular structure investigation. We apply LJ-PES assisted by electronic-structure calculations to study ATP solutions with and without dissolved Mg. Valence photoelectron data reveal spectral changes in the phosphate and adenine features of ATP due to interactions with the divalent cation. Chemical shifts in Mg 2p, Mg 2s, P 2p, and P 2s core-level spectra as a function of the Mg/ATP concentration ratio are correlated to the formation of [Mg(ATP) ], [MgATP], and [MgATP] complexes, demonstrating the element sensitivity of the technique to Mg-phosphate interactions. The most direct probe of the intermolecular interactions between ATP and Mg is delivered by the emerging ICD electrons following ionization of Mg 1s electrons. ICD spectra are shown to sensitively probe ligand exchange in the Mg-ATP coordination environment. In addition, we report and compare P 2s data from ATP and adenosine mono- and diphosphate (AMP and ADP, respectively) solutions, probing the electronic structure of the phosphate chain and the local environment of individual phosphate units in ATP. Our results provide a comprehensive view of the electronic structure of ATP and Mg-ATP complexes relevant to phosphorylation and dephosphorylation reactions that are central to bioenergetics in living organisms.
Topics: Magnesium; Adenosine Triphosphate; Photoelectron Spectroscopy
PubMed: 38802319
DOI: 10.1021/jacs.4c03174 -
Animal Nutrition (Zhongguo Xu Mu Shou... Jun 2024This study was to evaluate the effect of supplementing the diet of broilers with leaf extract (NCLE) on meat quality by evaluating antioxidant parameters and the...
This study was to evaluate the effect of supplementing the diet of broilers with leaf extract (NCLE) on meat quality by evaluating antioxidant parameters and the expression of genes in the p38 mitogen-activated protein kinase/nuclear factor-erythroid 2-related factor 2/antioxidant responsive element (p38 MAPK/Nrf2/ARE) signaling pathway, coupled with LC-MS-based metabolomic analysis. A total of 480 one-day-old male broilers were randomly allocated to four treatment groups-a control (CON) group, which was fed a basal diet, and three NCLE treatment groups, which were fed the basal diet supplemented with 100, 200, or 400 mg/kg NCLE (N1, N2, and N3 groups, respectively) for 42 d. Compared with the CON group, meat quality was improved in the N2 and N3 groups, as evidenced by the higher pH ( < 0.05) and lower shear force ( < 0.05) in breast muscle (BM) and lower drip loss at 48 h ( < 0.05) in leg muscle (LM). Moreover, BM antioxidant capacity was significantly enhanced in the N3 group, characterized by an increase in the total antioxidant capacity (T-AOC), the concentrations of glutathione peroxidase (GSH-Px) and catalase (CAT), and the relative mRNA expression of , extracellular-signal regulated kinase (), c-Jun N-terminal kinase (), , , and ( < 0.05). Similarly, LM in the N3 group displayed higher T-AOC, increased GSH-Px and CAT concentrations, reduced malonaldehyde contents ( < 0.05), and upregulation of the relative mRNA levels of , , heme oxygenase, , and superoxide dismutase () ( < 0.05). Metabolomics analysis revealed that D-arabinono-1,4-lactone and lyso-PAF C-16-d4 were negatively correlated with shear force and cooking loss ( < 0.05) and displayed increased abundance in BM of the N3 group. L-Serine levels were upregulated while D-fructose 1,6-diphosphate contents were downregulated in the three NCLE groups. Finally, the differential metabolites in both BM and LM were involved in amino acid metabolism pathways. Our results indicated that NCLE supplementation improved meat quality by enhancing antioxidant enzyme activities, promoting the expression of genes in the p38 MAPK/Nrf2/ARE signaling pathway, and regulating amino acid metabolism. The optimal NCLE concentration was found to be 400 mg/kg.
PubMed: 38800732
DOI: 10.1016/j.aninu.2024.01.011 -
Cureus Apr 2024Crigler-Najjar syndrome (CNS) is a genetic syndrome that results in increased levels of unconjugated bilirubin due to less or completely nonfunctional enzyme, uridine...
Crigler-Najjar syndrome (CNS) is a genetic syndrome that results in increased levels of unconjugated bilirubin due to less or completely nonfunctional enzyme, uridine diphosphoglucoronyltransferase (UDPGT) in hepatocytes. When bilirubin metabolism is compromised, hyperbilirubinemia is caused, which results in increased levels of unconjugated and conjugated bilirubin in the bloodstream. CNS is an autosomal recessive disorder, usually noticeable as people get older. This disorder is divided into two types: CNS type I and CNS type II, which are caused by homozygous or compound heterozygous mutations in the UDP glucuronosyltransferase family 1 member A1 (UGT1A1) gene. The disorder affects all races and genders equally, with a prevalence of one per million births. CNS type I is more severe and has almost undetectable UDPGT expression activity, and affected individuals die before one year of age. Consanguineous marriages are a major risk factor as CNS is inherited in an autosomal recessive manner. Being rare, maternal CNS type II is yet to be completely understood in terms of its impact on the mother, her pregnancy, and the infant. We aim to present a case of a pregnant female with CNS type II and its clinical course. She was monitored closely during her pregnancy. The treatment protocol was followed as per previously reported cases and was managed on low, non-teratogenic doses of phenobarbitone. A successful outcome with the birth of a healthy infant having normal neurological development till six months follow-up was observed.
PubMed: 38800243
DOI: 10.7759/cureus.59075