Structural and biochemical characterization of active sites mutant in human inorganic pyrophosphatase.

Inorganic pyrophosphatases (PPases) are enzymes that catalyze the conversion of inorganic pyrophosphate (PPi) into phosphate (Pi). Human inorganic pyrophosphatase 1 (Hu-PPase) exhibits high expression levels in a variety of tumors and plays roles in cell proliferation, apoptosis, invasion and metastasis, making it a promising prognostic biomarker and a target for cancer therapy. Despite its widespread presence, the catalytic mechanism of Hu-PPase in humans remains inadequately understood. The signature motif amino acid sequence (DXDPXD) within the active sites of PPases is preserved across different species. In this research, an enzymatic activity assay revealed that mutations led to a notable reduction in enzymatic function, although the impact of the four amino acids on the activity of the pocket varied. To investigate the influence of these residues on the substrate binding and enzymatic function of PPase, the crystal structure of the Hu-PPase-ED quadruple mutant (D116A/D118A/P119A/D121A) was determined at 1.69 Å resolution. The resulting structure maintained a barrel-like shape similar to that of the wild-type, albeit lacking Mg2+ ions. Molecular docking analysis demonstrated a decreased ability of Hu-PPase-ED to bind to PPi. Further, molecular dynamics simulation analysis indicated that the mutation rendered the loop of Mg2+ ion-binding residues less stable. Therefore, the effect on enzyme activity did not result from a change in the gross protein structure but rather from a mutation that abolished the Mg2+-coordinating groups, thereby eliminating Mg2+ binding and leading to the loss of enzyme activity.

Findings of ventilator-measured P0.1 in assessing respiratory drive in patients with severe ARDS.

BACKGROUND: Providers should adjust the depth of sedation to promote lung-protective ventilation in patients with severe ARDS. This recommendation was based on the assumption that the depth of sedation could be used to assess respiratory drive. OBJECTIVE: To assess the association between respiratory drive and sedation in patients with severe ARDS by using ventilator-measured P0.1 and RASS score. METHODS: Loss of spontaneous breathing was observed within 48 h of mechanical ventilation in patients with severe ARDS, and spontaneous breathing returned after 48 hours. P0.1 was measured by ventilator every 12 ± 2 hours, and the RASS score was measured synchronously. RESULTS: The RASS score was moderately correlated with P0.1 (R𝑆𝑝𝑒𝑎𝑟𝑚𝑎𝑛, 0.570; 95% CI, 0.475 to 0.637; p= 0.00). However, only patients with a RASS score of -5 were considered to have no excessive respiratory drive, but there was a risk for loss of spontaneous breathing. A P0.1 exceeding 3.5 cm H2O in patients with other RASS scores indicated an increase in respiratory drive. CONCLUSION: RASS score has little clinical significance in evaluating respiratory drive in severe ARDS. P0.1 should be evaluated by ventilator when adjusting the depth of sedation to promote lung-protective ventilation.

Astaxanthin encapsulation in soybean protein isolate-sodium alginate complexes-stabilized nanoemulsions: antioxidant activities, environmental stability, and in vitro digestibility.

BACKGROUND: Nanoemulsions (NEs) have been considered an effective carrier to protect environmentally labile bioactive compounds from degradation during food processing. Among the numerous types of NEs, biopolymer-stabilized NEs have gained much attention to achieve this function because of the extensive sources, biocompatibility, and tunability. Therefore, the antioxidant activities, environmental stability, and in vitro digestibility of astaxanthin (AST)-loaded soybean protein isolate (SPI)-alginate (SA) complexes-stabilized NEs (AST-SPI-SA-NEs) were investigated in this study. RESULTS: The AST-SPI-SA-NEs exhibited an encapsulation efficiency of 88.30 ± 1.67%, which is greater than that of the AST-loaded SPI-stabilized NEs (AST-SPI-NEs) (77.31 ± 0.83%). Both AST-SPI-SA-NEs and AST-SPI-NEs exhibited significantly stronger hydroxyl or diphenylpicryl-hydrazyl radical-scavenging activities than the free AST. The formation of SPI-SA complexes strengthened the thermal, light, and storage stability of AST-SPI-SA-NEs with no apparently increasing mean diameter (around 200 nm). AST-SPI-SA-NEs also exhibited a better freeze-thaw dispersibility behavior than AST-SPI-NEs. AST-SPI-SA-NEs were more stable than AST-SPI-NEs were under in vitro gastrointestinal digestion conditions and exhibited a greater bioaccessibility (47.92 ± 0.42%) than both AST-SPI-NEs (12.97 ± 1.33%) and free AST (7.87 ± 0.37%). Hydrogen bonding was confirmed to participate in the formation of AST-SPI-SA-NEs and AST-SPI-NEs based on the molecular docking results. CONCLUSIONS: The construction of SPI-SA-NEs is conducive to the encapsulation, protection, and absorption of AST, providing a promising method for broadening the application of AST in processed foods or developing novel ingredients of functional foods. © 2023 Society of Chemical Industry.

Mapping and Validation of a Stable Quantitative Trait Locus Conferring Maize Resistance to Gibberella Ear Rot.

Gibberella ear rot (GER), a prevalent disease caused by Fusarium graminearum, can result in significant yield loss and carcinogenic mycotoxin contamination in maize worldwide. However, only a few quantitative trait loci (QTLs) for GER resistance have been reported. In this study, we evaluated a Chinese recombinant inbred line (RIL) population comprising 204 lines, developed from a cross between a resistant parent DH4866 and a susceptible line T877, in three field trials under artificial inoculation with F. graminearum. The RIL population and their parents were genotyped with an Affymetrix microarray CGMB56K SNP Array. Based on the genetic linkage map constructed using 1,868 bins as markers, 11 QTLs, including five stable QTLs, were identified by individual environment analysis. Joint multiple environments analysis and epistatic interaction analysis revealed six additive and six epistatic (additive × additive) QTLs, respectively. None of the QTLs could explain more than 10% of phenotypic variation, suggesting that multiple minor-effect QTLs contributed to the genetic component of resistance to GER, and both additive and epistatic effects contributed to the genetic architecture of resistance to GER. A novel QTL, qGER4.09, with the largest effect, identified and validated using 588 F2 individuals, was colocalized with genomic regions for Fusarium ear rot and Aspergillus ear rot, indicating that this genetic locus likely confers resistance to multiple pathogens and can potentially be utilized in breeding maize varieties aimed at improving the resistance not only to GER but also other ear rot diseases.

Genome-Wide Characterization of Jasmonates Signaling Components Reveals the Essential Role of ZmCOI1a-ZmJAZ15 Action Module in Regulating Maize Immunity to Gibberella Stalk Rot.

Gibberella stalk rot (GSR) by Fusarium graminearum causes significant losses of maize production worldwide. Jasmonates (JAs) have been broadly known in regulating defense against pathogens through the homeostasis of active JAs and COI-JAZ-MYC function module. However, the functions of different molecular species of JAs and COI-JAZ-MYC module in maize interactions with Fusarium graminearum and regulation of diverse metabolites remain unknown. In this study, we found that exogenous application of MeJA strongly enhanced resistance to GSR. RNA-seq analysis showed that MeJA activated multiple genes in JA pathways, which prompted us to perform a genome-wide screening of key JA signaling components in maize. Yeast Two-Hybrid, Split-Luciferase, and Pull-down assays revealed that the JA functional and structural mimic coronatine (COR) functions as an essential ligand to trigger the interaction between ZmCOIa and ZmJAZ15. By deploying CRISPR-cas9 knockout and Mutator insertional mutants, we demonstrated that coi1a mutant is more resistant, whereas jaz15 mutant is more susceptible to GSR. Moreover, JA-deficient opr7-5opr8-2 mutant displayed enhanced resistance to GSR compared to wild type. Together, these results provide strong evidence that ZmJAZ15 plays a pivotal role, whereas ZmCOIa and endogenous JA itself might function as susceptibility factors, in maize immunity to GSR.