Enzyme Analysis Research Articles

Denitrifying microbial genes quantification attests inference for potential N2O emissions in sugarcane soils by enzymatic bioanalysis

This study evaluated the relationships and sensitivity of denitrifying microbial gene abundance, as well as the activities of soil enzymes β-glucosidase (GLU) and arylsulfatase (ARYL), to assess the quality of sugarcane soils managed with vinasse (V), filter cake (FC), and mineral fertilizer (MF). Composite soil samples were collected using a systematic sampling approach that included two soil classes (Ferralsol and Acrisol), two textures (clayey and sandy), three management systems (V, V+FC, and MF), two sampling seasons (rainy and dry), and three replicates, totaling 72 samples. Analysis of soil organic carbon (SOC), and macro- and micronutrients differentiated the Ferralsol and Acrisol samples into distinct groups based on agricultural management (Global R = 0.554) and showed some overlap based on soil texture (Global R = 0.369). The number of nirK, nirS, and nosZ I gene copies per gram of soil, determined by Real-Time Quantitative PCR (qPCR) based on genomic DNA isolated from the 72 soil samples, was higher in the rainy season compared to the dry season (P<0.05). None of the genes evaluated revealed a consistent response to different sugarcane soil managements, showing specific response patterns for each soil class and texture. In the Ferralsol, the activities of GLU and ARYL increased in the following order: V < MF < V+FC, regardless of soil texture (sandy or clayey) and sampling season. The average activity of the two enzymes in both V+FC and MF treatments was 1.8 times higher in sandy soil and 3.9 times higher in clayey soil compared to soil managed with vinasse. In the Acrisol, no significant differences among the treatments were observed. Statistical analyses revealed negative correlations (P<0.05) between the number of copies of the nirK and nosZ I genes and GLU and ARYL activities in the soil during both seasonal periods analyzed. The number of copies of these two microbial genes was also negatively correlated with the soil organic matter in the rainy season. Thus, the indications of sugarcane soil quality based on enzymatic analyses were corroborated by the lower abundance of genes associated with denitrification process. The findings of this study open the possibilities to infer about the potential for N2O emission from these sugarcane soils based on GLU and ARYL activities.

Genome-Wide Identification and Biochemical Characterization of Alcohol Acyltransferases for Aroma Generation in Wickerhamomyces subpelliculosus Isolates from Fermented Food.

The importance of nonconventional yeasts has increasingly been highlighted, particularly for aroma formation in fermented foods. Here, we performed de novo whole-genome sequencing of Wickerhamomyces subpelliculosus, which produces a variety of volatile flavor compounds, leading to the identification of the alcohol acyltransferase (AATase) family of genes. The genome of W. subpelliculosus contains seven AATase genes, encoding alcohol-O-acetyltransferases (ATFs) and ethanol acetyltransferase 1 (EAT1) for acetate ester formation, along with ethanol hexanoyl transferase 1 (EHT1) for ethyl ester formation. Among five ATF homologues, only WsATF5 showed acetyltransferase activity toward myriocin, a structural analogue of sphingosine. In contrast, heterologous expression of WsEHT1 and WsEAT1 in Saccharomyces cerevisiae promoted the production of ethyl decanoate and ethyl acetate, respectively, supporting their AATase activity. The enzymatic activity analyses revealed the additional alcoholysis activity of WsEAT1 and the thioesterase activity of WsEHT1. Subcellular localization analysis indicated that WsEAT1 was localized in the mitochondria, WsEHT1 in the endoplasmic reticulum and lipid droplets (LDs), and WsATF5 in the LDs. The novel W. subpelliculosus AATases could be usefully applied to produce flavor components in various food industries.

High-throughput Kinetics using capillary Electrophoresis and Robotics (HiKER) platform used to study T7, T3, and Sp6 RNA polymerase misincorporation

T7 RNA Polymerase (RNAP) is a widely used enzyme with recent applications in the production of RNA vaccines. For over 50 years denaturing sequencing gels have been used as key analysis tools for probing the nucleotide addition mechanisms of T7 RNAP and other polymerases. However, sequencing gels are low-throughput limiting their utility for comprehensive enzyme analysis. Here, we report the development of HiKER; (High-throughput Kinetics using Capillary Electrophoresis and Robotics) a high-throughput pipeline to quantitatively measure enzyme kinetics. We adapted a traditional polymerase misincorporation assay for fluorescent detection at scale allowing rapid estimates of RNAP misincorporation in different experimental conditions. Using this platform with an OT-2 robotics system, ~1500 time points were collected in a single workday. T7 RNAP exhibited dramatic differences in both observed rate constant and amplitude depending on the mismatch examined. An average misincorporation frequency of ~45 misincorporations per million bases was estimated using HiKER and is consistent with previous studies. Misincorporation time courses for T3 RNAP and Sp6 RNAP were also collected and appeared similar to T7 RNAP suggesting conserved kinetic mechanisms. However, differences between the RNAPs were observed in extension from base mismatch experiments. This platform is affordable, open-source, and broadly applicable to many enzymes.

Characterization and synergistic activity of heterologously expressed microbial-derived endoglucanase and bifunctional cellulase on wheat straw

Cellulases are divided into endoglucanase, exoglucanase, and β-glucosidase based on their catalytic activity. Eight cellulases were recombinantly expressed in Escherichia coli BL21 to investigate their effects on the enzymatic hydrolysis of wheat straw. Among them, cellulase 2006 exhibited the highest endoglucanase activity (432.25 U/mg), while Bf1 displayed superior exoglucanase and β-glucosidase activities (577.46 and 1991.63 U/mg respectively). Bioinformatic and enzymatic analyses revealed that both cellulases displayed notable thermal and pH stability. The enzyme kinetics parameters revealed that Km values for cellulases 2006 and Bf1 were 15.98 and 14.19 mg/mL, respectively, with Vmax values of 20.78 and 16.38 μmol/min/mg. In a prokaryotic co-expression system, the mixed cellulase Bf2006 exhibited endoglucanase, exoglucanase, and β-glucosidase activities (130.78, 1406.36, and 1119.25 U/mg, respectively). The enzymatic hydrolysis assay revealed that these three cellulases acted on the cellulose macromolecules in wheat straw, increasing reducing sugar content and decreasing crystallinity. Endoglucanase 2006 acted on various organic compounds rich in phenols and aromatic heterocycles, while Bf1 primarily acted on compounds containing glucose units. Bf2006 significantly affected the content of lignin, neutral detergent fiber, acid detergent fiber, and the microstructure of wheat straw, with degradation products primarily consisting of disaccharides, oligosaccharides, polysaccharides, glycosides, and other carbohydrates. This study provides theoretical guidance for the production and application of mixed cellulase Bf2006, serving as a reference for the industrial use of different cellulase types.

Structure-guided engineering of 4-coumarate: CoA ligase for efficient production of rosmarinic acid in Saccharomyces cerevisiae

The utilization of genetically modified microbial cells for rosmarinic acid (RA) production is gaining increased attention as a cost-effective and sustainable approach. However, the substrate promiscuity of 4-coumarate: CoA ligase and RA synthase has been considered as a critical factor for low RA yields. In this study, we rationally engineered the substrate preference of 4-coumarate: CoA ligase (OPc4CL2) from Petroselinum crispum, resulting in a significant enhancement in RA production. Particularly, the introduction of the Y240C mutation led to a remarkable 176% increase in RA yield. Subsequent enzymatic analysis of OPc4CL2 variants revealed diminished activity towards p-coumaric acid, resulting in insufficient time for the transformation of p-coumaric acid to 4-coumaroyl CoA to generate byproduct. Furthermore, to minimize the formation of undesired byproducts, the overexpression of 4-hydroxyphenylacetate 3-monooxygenase (OHpaB) and NADPH-flavin oxidoreductase (HpaC) was carried out to facilitate the conversion of p-coumaric acid to caffeic acid and 4-hydroxyphenyllactate to salvianic acid A, thus achieving a significant increase in RA yield of up to 329.9mg/L (16.5mg/g yield on glucose) in shake-flask cultivation. Finally, the engineered strain YRA113-24BHM achieved a notable RA production of 3.6g/L (about 20.2mg/g yield on glucose) by fed-batch fermentation. This study serves as a foundation for the sustainable biosynthesis of RA and other caffeic acid derivatives.

Impact of right-sided breast cancer adjuvant radiotherapy on the liver.

In patients with right-sided breast cancer the liver can be partially irradiated during adjuvant radiotherapy (RT). We aimed to determine breast cancer RT effects on liver using with magnetic resonance elastography (MRE) and biological results. This retrospective study enrolled 34 patients diagnosed with right-sided breast cancer who underwent adjuvant RT. Liver segment assessments were conducted using MRE for all participants. Additionally, a complete blood count and liver enzyme analysis were performed for each patient. All measurements were taken both prior to the initiation and upon completion of RT. A statistically significant difference was found in ALT (p = 0.015), ALP (p = 0.026), total protein (p = 0.037), and albumin (p = 0.004) levels before and after RT. The highest mean liver stiffness (kPa) value was recorded in segment 8, while the lowest was observed in segment 6. A weak but statistically significant positive correlation was found between segment 5 stiffness and liver volume (p = 0.039). Additionally, a statistically significant positive correlation was detected between ALP levels and the stiffness values in segment 4A (p = 0.020) and segment 6 (p = 0.003). Conversely, a weak negative correlation was observed between the stiffness values in segment 8 and post-RT total protein levels (p = 0.031). MRE can help us identify the level of fibrotic stiffness in the liver segments within the RT area without establishing clinical symptoms. MRE can support the clinician in evaluating the liver functions of right breast cancer patients who underwent RT. We assume these results will facilitate new studies with a large number of patients on MRE imaging at certain intervals in the follow-up of patients with right breast cancer who received RT before the development of radiation-induced liver disease (RILD).

Exploration of urease-aided calcium carbonate mineralization by enzyme analyses of Neobacillus mesonae strain NS-6.

Urease containing nickel cofactor is crucial for urea-hydrolytic induced calcium carbonate (CaCO3) precipitation (UICP). However, limited information exists regarding the influence of amino acid residues interacting with nickel ions in its structure on induced CaCO3 mineralization. Herein, RT-qPCR was used to demonstrate that the addition of NiCl2 dramatically upregulated the expression of urease structural gene ureC that was correlated with nickel binding in Neobacillus mesonae strain NS-6. Homology modeling and molecular docking were employed to construct the three-dimensional structure of urease and seek the key residues involved in nickel binding process, and virtual mutation technology was adopted to inform three key residues coordinated with nickel ions and urea, His249, His275, and Asp363. Four metrics, including root mean square deviation values for mutations of those key residues in urease-urea complexes severally and wild-type, were calculated by molecular dynamics simulations when they were mutated into alanine, respectively. Subsequently, the mutations of H249A, H275A, and D363A were characterized using western blotting to reveal a decrease in the relative expression and activity of urease, along with a corresponding reduction in CaCO3 precipitation. Ultimately, the mutations also exhibited that they had lower substrate affinity and catalytic efficiency for urea through enzymatic properties analysis. The findings suggested that those residues played a pivotal role in UICP of strain NS-6, which would expand the theoretical basis for modulating urease activity.IMPORTANCEUrease-producing bacterium is of great importance in diverse application fields, such as environmental remediation, due to its key driving characteristics in catalyzing urea hydrolysis via urea-hydrolytic induced CaCO3 precipitation (UICP). As essential cofactors of urease, nickel ions play a crucial role in regulating urease catalysis and maintaining structural stability. Numerous investigations have emphasized the impact of nickel ions on urease activity in recent years, to our best knowledge, only a few literatures have studied the molecular-level regulation of nickel-ligand residues. This study focused on the highly urease-producing bacterial Neobacillus mesonae NS-6 to explore the effects of specific nickel-ligand residues on the urease-aided CaCO3 mineralization process using molecular simulation predictions and targeted mutation experiments. The aim was to provide a molecular-level understanding of the interactive effects between urea and critical residues associated with the urease active center, as well as propose an effective modification strategy to enhance the application of UICP in future environmental areas.

Sulfuric acid effects in seed dormancy break of Ipomoea batatas evaluated by enzymatic, anatomic and physiological approaches

ABSTRACT The recommended method to overcome seed coat dormancy in sweet potato seeds is exposure to sulfuric acid, which can damage the embryo and affect seedling development depending on exposure time. This study aimed to determine the ideal immersion period using physiological, anatomical, and enzymatic analyses. Seeds were scarified with concentrated sulfuric acid for nine immersion periods. Physiological analyses included germination tests, germination speed index, first germination count, emergence test, and emergence speed index. Image analysis quantified the primary root length, root-to-hypocotyl ratio, and seedling length. Catalase and superoxide dismutase enzyme activities were analysed. Seed coat anatomy, including thickness, wear, and colour, was also assessed. Exposure to sulfuric acid for 30 min effectively broke dormancy without impacting the antioxidant system or seedling development. Thermal chambers confirmed that chemically scarified seeds absorb water and activate metabolism faster. Plant anatomy helped illustrate how sulfuric acid breaks dormancy by eroding physical barriers.

LINE-1 hypomethylation characterizes the inflammatory response in coeliac disease associated-intestinal mucosa and small bowel adenocarcinomas.

Long interspersed nuclear elements 1 (LINE-1) are the most abundant and the only autonomous mobile elements in the human genome. When their epigenetic repression is removed, it can lead to disease, such as autoimmune diseases and cancer. Coeliac disease (CeD) is an immune-mediated disease triggered by an abnormal T-cell response to dietary gluten and a predisposing condition of small bowel adenocarcinoma (SBA), frequently characterized by epigenetic alterations. The aim of this work was to assess LINE-1 methylation by bisulphite pyrosequencing and NanoString® gene transcription analysis in 38 CeD-SBAs compared with 25 SBAs associated with Crohn's disease (CrD-SBAs) and 25 sporadic SBAs (S-SBA). Both analyses were also performed in duodenal mucosae from 12 untreated CeD patients (UCD) and 19 treated CeD patients (TCD), and in 11 samples of normal intestinal mucosa to better investigate the role of LINE-1 deregulation in CeD and in CeD-SBA. A significant loss of LINE-1 methylation was observed in CeD-SBAs and in mucosae from UCD patients (with very similar methylation levels) compared with controls. By contrast, a restoration of normal LINE-1 methylation levels was found in TCD mucosae after a strict gluten-free diet. LINE-1 hypomethylation does not lead to expression of ORF1 and ORF2, with the only exception being for one CeD-SBA. The expression analysis of enzymes modulating DNA methylation and inflammatory genes confirmed that CeD-SBA shared a very similar expression profile of UCD mucosae showing a strong upregulation of genes involved in inflammation, immune response, and T-cell activity compared with TCD mucosae. For the first time, this work demonstrates that loss of DNA methylation is an intrinsic epigenetic feature of CeD, accompanying the immune response as a reversible mechanism in patients following a strict gluten-free diet, and suggests the possible role of LINE-1 hypomethylation in promoting cell adaptability during the gliadin-related inflammatory process. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Precision Activity-Based α-Amylase Probes for Dissection and Annotation of Linear and Branched-Chain Starch-Degrading Enzymes.

α-Amylases are the workhorse enzymes of starch degradation. They are central to human health, including as targets for anti-diabetic compounds, but are also the key enzymes in the industrial processing of starch for biofuels, corn syrups, brewing and detergents. Dissection of the activity, specificity and stability of α-amylases is crucial to understanding their biology and allowing their exploitation. Yet, functional characterization lags behind DNA sequencing and genomics; and new tools are required for rapid analysis of α-amylase function. Here, we design, synthesize and apply new branched α-amylase activity-based probes. Using both α-1,6 branched and unbranched α-1,4 maltobiose activity-based probes we were able to explore the stability and substrate specificity of both a panel of human gut microbial α-amylases and a panel of industrially relevant α-amylases. We also demonstrate how we can detect and annotate the substrate specificity of α-amylases in the complex cell lysate of both a prominent gut microbe and a diverse compost sample by in-gel fluorescence and mass spectrometry. A toolbox of starch-active activity-based probes will enable rapid functional dissection of α-amylases. We envisage activity-based probes contributing to better selection and engineering of enzymes for industrial application as well as fundamental analysis of enzymes in human health.

TDO2 inhibition counters Benzo[a]pyrene-induced immune evasion and suppresses tumorigenesis in lung adenocarcinoma

IntroductionBenzo[a]pyrene (BaP) is a toxic polycyclic aromatic hydrocarbon known as an exogenous AhR ligand. This study investigates the role of BaP in inducing immune checkpoint expression in lung adenocarcinoma (LUAD) and the underlying mechanisms involving the aryl hydrocarbon receptor (AhR) and tryptophan (Trp) metabolism.MethodsWe assessed the expression of immune checkpoint molecules, including PD-L1 and ICOSL, in lung epithelial cell lines (BEAS-2B and H1975) exposed to BaP. The involvement of AhR in BaP-induced immune checkpoint expression was examined using AhR silencing (siAhR). Additionally, the role of Trp metabolism in BaP-mediated immune evasion was explored through culturing in Trp (-/+) condition media, treatments with the inhibitors of rate-limiting enzymes in Trp metabolism (TDO2 and IDO1) and analyses of Trp-catabolizing enzymes. The therapeutic potential of targeting Trp metabolism, specifically TDO2, was evaluated in vivo using C57BL/6 mice orthotopically inoculated with LUAD cells.ResultsBaP exposure significantly upregulated the mRNA and surface expression of PD-L1 and ICOSL, with AhR playing a crucial role in this induction. Trp metabolism was found to enhance BaP-mediated immune evasion, as indicated by stronger induction of immune checkpoints in Trp (+) media and the upregulation of Trp-catabolizing enzymes. TDO2 inhibition markedly suppressed the surface expression of PD-L1 and ICOSL, demonstrating the importance of Trp metabolism in BaP-induced immune evasion. Further analysis confirmed the high TDO2 expression in lung adenocarcinoma and its association with poor patient survival. Using an orthotopic implantation mouse model, we demonstrated the inhibitory effect of two different TDO2 inhibitors on tumorigenesis, immune checkpoints, and tryptophan metabolism.ConclusionsThis study highlights the key mechanisms behind BaP-induced immune evasion in LUAD, particularly through the TDO2/AhR axis. It reveals how TDO2 inhibitors can counteract immune checkpoint activation and boost anti-tumor immunity, suggesting new paths for targeted lung cancer immunotherapy. The findings significantly improve our understanding of immune evasion in LUAD and underscore the therapeutic promise of TDO2 inhibition.

Catalytic mechanism underlying the regiospecificity of coumarin-substrate transmembrane prenyltransferases in Apiaceae.

Plant membrane-bound prenyltransferases (PTs) catalyse the transfer of prenyl groups to acceptor substrates, phenols, using prenyl diphosphates as the donor substrate. The presence of prenyl residues in the reaction products, prenylated phenols, is key to the expression of a variety of physiological activities. Plant PTs generally exhibit high specificities for both substrate recognition and prenylation sites, while the molecular mechanism involved in these enzymatic properties is largely unknown. In this study, we performed a systematic biochemical analysis to elucidate the catalytic mechanism responsible for the reaction specificity of plant PTs. Using two representative PTs, PsPT1 and PsPT2, from parsnip (Pastinaca sativa, Apiaceae), which differ only in the regiospecificity of the prenylation site, we performed domain swapping and site-directed mutagenesis of these PTs, followed by detailed enzymatic analysis combined with three-dimensional modelling. As a result, we discovered the domains that control prenylation site specificity and further defined key amino acid residues responsible for the catalytic mechanism. In addition, we showed that the control mechanism of prenylation specificity revealed here is also highly conserved among coumarin-substrate PTs. These data suggest that the regulatory domain revealed here is commonly involved in prenylation regiospecificity in Apiaceae PTs.

Systemic acquired resistance inducing chemicals mitigate black scurf disease in potato by activating defense-related enzymes.

The potato, being an underground vegetable crop, faces consistent threats from soil- and tuber-borne fungal and bacterial pathogens. Black scurf and stem canker disease caused by the fungal pathogen Rhizoctonia solani Kuhn is a critical global concern in the potato cultivation system. In this study, we evaluated the disease mitigation potential of five systemic acquired resistance-inducing chemicals viz., salicylic acid, jasmonic acid, β-aminobutyric acid, γ-aminobutyric acid and hydrogen peroxide (H2O2). Two common methods, tuber dipping and foliar spray, were utilized in this experiment to evaluate pathogen inhibition on inoculated tubers. The results revealed that all the systemic acquired resistance inducing chemicals were effective in disease suppression in a concentration-dependent manner compared to an inoculated control. Significant differences (P<0.005) were evident among the various treatment combinations, with salicylic acid being the most effective in alleviating black scurf disease. Maximum reduction in disease incidence compared to the control was observed with salicylic acid (57.89% and 73.68%), followed by jasmonic acid (52.63% and 65.78%) and H2O2 (49.99% and 60.52%) under the tuber dipping treatment combinations. Whereas, in the foliar application, the maximum reduction in disease incidence compared to the control was observed with salicylic acid (44.73 and 63.15%), followed by jasmonic acid (42.10 and 60.52%) and H2O2 (39.46 and 52.63%). The tuber dipping treatments were significantly more efficacious (P<0.005) compared to foliar spray for all treatment combinations. The biochemical analysis of defense-related enzymes and metabolites demonstrated the induced resistance activation under these treatments. The activity of peroxidase, polyphenol oxidase, and phenyl ammonia-lyase was significantly higher in treated tubers as compared to inoculated and uninoculated control. The total phenol content was also elevated in treated tubers as compared to the respective control. Altogether, these resistance-inducing chemicals can be successfully included in integrated disease management programs.

Role of phenylpropanoid pathway in genetic regulation and physiological adaptation in arsenic stressed rice genotypes

This study investigates the role of the phenylpropanoid pathway in arsenic (As) contaminated rice genotypes under natural conditions, exploring the intricate relationship between genetic regulation and physiological adaptation. Differential approaches adapted by rice genotypes to counteract As exposure are elucidated here through analysis of enzyme activities and related gene expression patterns, docking simulations, and nutrient dynamics. Enzymatic analysis from the phenylpropanoid pathway revealed significant variations across rice genotypes, with Mini mansoori exhibiting notably higher activity levels of key enzymes (PAL, C4H, 4CL, CHI, DFR and F3H) compared to Sampoorna and Pioneer. Additionally, the gene expression profiling unveiled differential responses, with Mini mansoori and Pioneer demonstrating higher expression of genes (OsPAL, OsCHS, OsCHI, OsF3H, OsF3′H, OsFLS, OsDFR, and OsLAR) associated with As resistance and tolerance, compared to Sampoorna. Enrichment analysis emphasized the involvement of cinnamic acid biosynthesis and related pathways. Molecular docking depicted certain proteins, such as Os4CL, OsFLS, OsDFR, and OsLAR susceptible to As binding, potentially affecting enzymatic activity. Ionomic analysis unveiled that Mini mansoori maintained higher levels of essential nutrients such as Na, Ca, P, Mn, Mg, and Zn in grains. However, this contrasted with Pioneer and Sampoorna, which experienced nutrient imbalance likely due to higher As accumulation. Chlorophyll fluorescence analysis depicted that Mini mansoori and Pioneer maintained better photosynthetic efficiency under As toxicity compared to Sampoorna. Moreover, network analysis highlights the critical role of Mg and Na interaction with essential phenolics and flavonoids, in combating the stress. Harnessing this understanding, targeted breeding effort could yield As-resistant rice varieties with enhanced nutrient and flavonoid contents, addressing both food safety and malnutrition in affected regions.

Longissimus from Berkshire pigs in a small-scale supply chain have increased oxidative metabolism, tenderness and water-holding capacity, compared with Large White × Landrace pigs in a modern commercial supply chain

Context Selection for leanness in the modern Australian pig has resulted in inconsistent quality, including a lack of pork tenderisation during ageing. Inconsistent quality is potentially a result of differences in supply chain and breed as well as the variation in muscle fibre-type proportion in pork longissimus. Aim The aim was to investigate differences in fibre-type proportion and pork quality between Large White-Landrace pigs in a large supply chain and Berkshire pigs processed in a small supply chain. Methods Pigs (n = 22) from two suppliers with different breeds (Supplier 1, Large White × Landrace, SC1-LWLR, n = 12, modern commercial pigs; Supplier 2, Berkshire, SC2-Berk, n = 10, heritage pigs) were slaughtered and samples from the longissimus were extracted at 3, 24, and 48 h postmortem for enzyme and pH analyses. Longissimus samples were subjected to ageing for either 2 or 16 days postmortem (Day 2, Day 16), assessed for colour, muscle fibre-type proportion (%), muscle fibre diameter (μm), water-holding capacity (purge, % and cook loss, %), Warner–Bratzler peak shear force (WBSF, N), and protein denaturation temperature using differential scanning calorimetry (DSC, peak temperature, °C). Key results SC1-LWLR had higher purge than SC2-Berk (2.85% and 1.83% respectively; standard error of the difference (SED) = 0.33; P = 0.003), higher cook loss on Day 16 (24.63% and 16.79% respectively; SED = 1.62; P = 0.017) and higher WBSF on Day 2 and Day 16 (Day 2, 30.9 N and 26.7 N respectively; Day 16, 28.6 N and 22.0 N respectively; SED = 0.98, interaction P = 0.003). SC1-LWLR had a lower proportion of Type I (10.1% vs 16.0%; SED = 0.51) and Type IIA (14.0% vs 22.0%; SED = 0.77) and a higher proportion of Type IIB (75.9% vs 62.0%; SED = 0.74) (P &lt; 0.001 for all) fibres. SC1-LWLR had lower DSC temperatures for two peaks. SC2-Berk had higher citrate synthase activity (P = 0.003) and glycolytic potential (P &lt; 0.001) than SC1-LWLR. Conclusions SC2-Berk longissimus had improved quality compared with SC1-LWLR pork, most likely owing, in part, to higher proportion of oxidative and intermediate fibres in the Berkshires. However, effects of differences in environmental conditions and/or processing conditions cannot be ruled out. Implications The experiment increased our understanding of how variation in supply chains and muscle fibre-type proportion can impact the production of consistently high-quality pork.

Enhancing Molecular‐Level Biological Monitoring with a Smart Self‐Assembling 19F‐Labeled Probe

AbstractReal‐time monitoring of molecular transformations is crucial for advancements in biotechnology. In this study, we introduce a novel self‐assembling 19F‐labeled nuclear magnetic resonance (NMR) probe that disassembles upon interaction with various nucleotides. This interaction not only activates the 19F signals but also produces distinct signatures for each specific component, thereby enabling precise identification and quantification of molecules in evolving samples. We demonstrate the capability of this probe for real‐time monitoring of adenosine triphosphate (ATP) hydrolysis and screening potential enzyme inhibitors. These applications highlight the probe's significant potential in enzyme analysis, drug development, and disease diagnostics.

Enhancing Molecular-Level Biological Monitoring with a Smart Self-Assembling 19F-Labeled Probe.

Real-time monitoring of molecular transformations is crucial for advancements in biotechnology. In this study, we introduce a novel self-assembling 19F-labeled nuclear magnetic resonance (NMR) probe that disassembles upon interaction with various nucleotides. This interaction not only activates the 19F signals but also produces distinct signatures for each specific component, thereby enabling precise identification and quantification of molecules in evolving samples. We demonstrate the capability of this probe for real-time monitoring of adenosine triphosphate (ATP) hydrolysis and screening potential enzyme inhibitors. These applications highlight the probe's significant potential in enzyme analysis, drug development, and disease diagnostics.

Metallomic Approach to Mercury and Selenium in the Liver Tissue of Psectrogaster amazonica and Raphiodon vulpinus from the Brazilian Amazon.

This paper reports the results of a mercury (Hg) and selenium (Se) metallomic study in the liver tissues of Psectrogaster amazonica and Raphiodon vulpinus from the Brazilian Amazon. Two-dimensional electrophoresis, graphite furnace atomic absorption spectrometry, and liquid chromatography-tandem mass spectrometry were performed. Hg and Se determinations allowed the calculation of Hg:Se and Se:Hg molar ratio and Se values for health benefits (Se HBVs). The Se:Hg values were >1 for both fish species, whereas the Se HBVs were >5 for P. amazonica and >10 for R. vulpinus, indicating that both possess Se reserves to control Hg toxicity. The metallomic data allowed the identification of 11 Hg/Se-associated protein spots in the two fish species, with concentrations in the range of 9.70 ± 0.14 and 28.44 ± 0.31 mg kg-1 of Hg and 16.15 ± 0.21 and 43.12 ± 0.51 mg kg-1 of Se. Five metal binding proteins (MBP) in the Hg/Se-associated protein spots in the liver proteome of P. amazonica and eight in R. vulpinus were identified, indicating the possible formation of Hg/Se complexes on the MBP structures. The activities analysis of catalase, superoxide dismutase, GPx enzymes, and lipoperoxide concentrations demonstrated that Hg-induced oxidative stress did not occur, possibly because both fish species possess Se reserves necessary to inhibit the Hg's deleterious effects.

Functional analysis of a S-adenosylmethionine-insensitive methylenetetrahydrofolate reductase identified in methionine-accumulating yeast mutants.

Essential amino acids (EAAs) are important for the maintenance of brain functions. Therefore, the yeast Saccharomyces cerevisiae that accumulate EAAs would help elderly people ingest appropriate levels of EAAs, which in turn could slow neurodegeneration, extend the healthy lifespan and improve quality of life. Here, we isolated two mutant strains, ETH-80 and ETH-129, that accumulate the EAA methionine. Both strains were derived from a diploid laboratory yeast by conventional mutagenesis and carry a novel mutation in the MET13 gene, which encodes the Ser443Phe variant of methylenetetrahydrofolate reductase. Enzymatic analysis revealed that the Ser443Phe substitution abolished the sensitivity to S-adenosyl methionine (SAM)-mediated inhibition even in the presence of 2mM SAM, while increasing the activity for NADPH-dependent reduction. Furthermore, yeast cells expressing the Ser443Phe variant showed a fourfold increase in intracellular methionine content compared to the wild-type Met13. These findings will be useful for the future development of methionine-accumulating yeast strains.

Evaluation of the Safety and Efficacy of Curcumin-Synthesized Silver Nanoparticles in Rats Exposed to Chlorpyrifos During Puberty Development.

Silver nanoparticles (Ag-NPs) have garnered significant attention in recent years due to their therapeutic effects. Curcumin (CUR) has been utilized as a coating agent for synthesizing Ag-NPs, intended to act as a potential drug. This study was designed to evaluate the safety and efficacy of curcuminsynthesized silver nanoparticles on rats exposed to chlorpyrifos (CPF) during their pubertal development. Forty-two male Wistar rats, 23 days old, were selected and randomly divided into 7 groups (n=6) as follows: positive control, negative control, CPF (5 mg/kg), silver nanoparticles synthesized using curcumin at 40 μg/kg (CUR-Ag-NPs 40), CUR-Ag-NPs 80, CPF+ CUR-Ag-NPs 40, CPF+ CUR-AgNPs 80. All treatments were administered via gavage for 30 days. At the end of the study, rats were anesthetized using ketamine (50 mg/kg), and xylazine, (10 mg/kg) and blood was collected from the heart for serum analysis of liver enzymes, urea, and creatinine. Liver and kidney tissues were isolated for histopathological analysis. No significant differences were observed in serum levels of AST, ALT, and ALP enzymes as well as urea and creatinine levels among the different groups. Light microscopy observation revealed multifocal inflammatory mononuclear cell subsets in liver tissue associated with mild inflammatory mononuclear cell infiltration in the portal region in CPF, CUR-Ag-NPs 40, CUR-Ag-NPs 80, CPF+CUR-Ag-NPs 40, and CPF+CUR-Ag- NPs 80 groups. Histological examination of kidney tissue showed degenerative changes in the tubular epithelium, congestion, and mild infiltration of mononuclear inflammatory cells in the renal interstitial tissue in the CPF group, CUR-Ag-NPs 40, CUR-Ag-NPs 80, CPF+CUR-Ag-NPs 40 and CPF+CUR-Ag-NPs 80 groups. This study failed to establish the safety and efficacy of CUR-Ag-NP at 40 and 80 μg/kg in prepubertal rats exposed to CPF. However, further studies should be conducted to thoroughly characterize the efficacy of CUR-Ag-NP in developmental animal models.