Altered Enzyme Activity Research Articles

Colonization of phthalate-degrading endophytic bacterial consortium altered bacterial community and enzyme activity in plants

Phthalates (PAEs) are widely distributed hazardous organic compounds that pose threats to ecosystems and human health. Endophytic bacteria can effectively eliminate PAEs contamination risk. However, limited information is available regarding the impact of endophytic bacterial colonization on bacterial communities within plants. In this study, the endophytic bacterial consortium EN was colonized in lettuce by seed soaking, root irrigation, leaf spraying, and combined spraying-irrigation, resulting in a marked improvement in plant growth. The findings revealed that consortium EN colonization through combined spraying-irrigation exhibited superior degradation capability with 40.54% PAEs removal from soil. Meanwhile, the residual PAEs in lettuce decreased by 94.05% compared with the uninoculated treatment. High-throughput sequencing analysis indicated that colonization of consortium EN altered the bacterial community in lettuce. Specifically, the relative abundance of the dominant genus Pseudomonas was significantly higher than that in the uninoculated control (P < 0.01). Additionally, colonization enhanced the activities of peroxidase and catalase in lettuce, thereby improving plant resistance. This work offers a theoretical foundation for comprehending the mechanism underlying the bioremediation of PAEs contamination by endophytic bacteria in soil-plant system.

Testicular enzyme activity alterations in rats with liver cirrhosis induced by alcohol and acetaminophen

Alcohol-induced acetaminophen (APAP) toxicity is one of the numerous factors that might result in liver cirrhosis (LC). The hepatotoxicity of APAP appears to increase in chronic drinkers, according to a number of investigations. These people not only have a greater risk of experiencing acute overdose-related severe and deadly liver damage but are also at risk of experiencing similar substantial liver damage from therapeutic APAP use. The male reproductive system consists of both testes and a few other auxiliary sexual organs. Smaller and lighter testicles are indicative of severe cirrhosis causing testicular atrophy. In the case of severe LC, low testosterone results from hypogonadotropic hypogonadism. Sexual dysfunction is a prevalent condition that is frequently overlooked in individuals suffering from cirrhosis and chronic liver disease. Eighteen rats were randomly divided into three groups. Rats of normal control group received water and normal diet ad libitum; alcohol control and LC group received 4.5% alcohol and a combination of 4.5% alcohol and APAP (300 mg/kg bw) through drinking water, respectively, for 7 days. Several glycolytic and antioxidant enzymes were assessed for their effects through non-denaturing polyacrylamide gel electrophoresis analysis and enzyme activity. The results indicated that long-term alcohol consumption and APAP medication altered the levels of antioxidant and glycolytic enzymes. To the best of our knowledge, this is the first study demonstrating that chronic alcoholism and APAP induce hepatotoxicity, and LC further affects the antioxidant and glycolytic enzyme activities of the testes.

Frailty and sarcopenia metabolomic signatures in kidney transplant candidates: the frailmar study

Abstract Sarcopenia and frailty are often overlooked in assessing kidney transplant (KT) candidates with chronic kidney disease (CKD), potentially leading to poor post-transplant outcomes. This study aimed to identify metabolites associated with frailty and sarcopenia in KT-candidates from the FRAILMAR study. Between June 2016 and June 2020, we evaluated frailty and sarcopenia in 173 KT-candidates using the Physical Frailty Phenotype and EGWSOP-2 criteria, respectively. Seventy-five metabolic markers from targeted pathways, previously linked to CKD, sarcopenia, or frailty, were measured in serum samples. These markers were analyzed using adjusted and weighted generalized linear models. Metabolomic data were integrated with multi-modal data, such as comorbidities, using a factor-based integration algorithm to identify metabolic phenotypes. Increased metabolites related to energy metabolism and essential amino acids were associated with frailty, mainly Krebs cycle intermediates. Sarcopenic KT-candidates showed lower levels of aromatic amino acids, lower protein/muscle metabolism, energy metabolism, and neurotransmission compared to non-sarcopenic patients. Unsupervised multi-modal integration revealed a high-risk metabolic phenotype characterized by the presence of sarcopenia, diabetes mellitus, and low body mass index, with alterations in branched-chain amino acids and high activity of lactate dehydrogenase enzyme. Frailty and sarcopenia are common among KT-candidates, and their metabolic status reveal notable disruptions in energy and amino acid metabolism. These findings highlight the value of a detailed metabolic assessment to more accurately evaluate patient health status prior to transplantation.

Synergistic effects of thermal stress and 4-nonylphenol on oxidative stress and immune responses in juvenile tilapia (Oreochromis niloticus).

Aquatic ecosystems face multiple stressors, including thermal fluctuations and chemical pollutants, which can detrimentally impact fish health and ecosystem integrity. This study investigates the individual and combined toxic effects of 4-nonylphenol (4-NP) and thermal stress on juvenile tilapia fish (Oreochromis niloticus). Four groups of fish were exposed to different stressors for 15days: control, thermal stress (35°C ± 1°C), 4-NP exposure (1mg/L), and a combination of thermal stress and 4-NP. Results reveal significant alterations in antioxidant enzyme activity, lipid peroxidation levels, and cytokine expression in response to stressors. Thermal stress and 4-NP exposure disrupt antioxidant defense mechanisms and increase oxidative stress. Thermal stress profoundly affects fish health and metabolism, impacting physiological functions and immunity. Thermal stress induces reactive oxygen species production, triggering antioxidant responses and affecting immune parameters. Exposure to 4-NP exacerbates oxidative stress, further compromising fish health. The observed increase in pro-inflammatory cytokines implies an immunostimulatory reaction to stressors. These findings underscore the complex interactions between environmental stressors, immune responses, and fish health. Further research is needed to fully understand these interactions and their implications for aquatic ecosystems. Implementing these biomarkers in ecological risk assessments can provide insights into the impacts of environmental stressors and inform conservation and management strategies in aquaculture.

Comparative study of fenpropathrin and its main metabolite in soil-earthworm microcosms: Toxicity, degradation, transcriptome, and oxidative stress

This study comprehensively investigated the comparative acute toxicities, degradation, transcriptome, and oxidative stress induction of fenpropathrin (FEN) and its main metabolite 3-phenoxybenzoic acid (3-PBA)in soil-earthworm microcosms. FEN degradation half-life ranged from 19.09 to 28.52 days, and the peak-shaped trends of 3-PBA were also observed in different soil types. The LC50 values of FEN and 3-PBA were 12.75 and 7.49 μg/cm2, respectively, suggesting that 3-PBA was more toxic to earthworms. Furthermore, the sub-lethal toxicities indicated that 3-PBA exerted more prominent alterations in protein content, enzyme activity, lipid peroxidation, and oxidative stress in earthworms. Additionally, integrated biomarker response evaluations indicated that 3-PBA induced more prominent sub-lethal toxicity in earthworms than FEN. Finally, exposure to FEN and 3-PBA resulted in distinct differentially expressed genes (DEGs) in earthworms. Enrichment analysis revealed that these DEGs were predominantly enriched in purine metabolism and bile secretion pathways in earthworms. Moreover, the p53 signaling pathway, cell cycle, DNA replication, drug metabolism, and pyrimidine metabolism were also enriched in earthworms after exposure to FEN and 3-PBA. These results suggested that FEN and 3-PBA induced varying toxicities in earthworms. This study highlighted the systemic differences in the toxicities, degradation, transcriptome, and oxidative stress induction between FEN and 3-PBA in soil-earthworm microcosms. Our findings could be used for a comprehensive risk assessment of FEN and 3-PBA in the soil ecosystem.

Changes in Plasma Clearance of CYP450 Probe Drugs May Not be Specific for Altered In Vivo Enzyme Activity Under (Patho)Physiological Conditions: How to Interpret Findings of Probe Cocktail Studies.

CYP450 (CYP) phenotyping involves quantifying an individual's plasma clearance of CYP-specific probe drugs, as a proxy for in vivo CYP enzyme activity. It is increasingly applied to study alterations in CYP enzyme activity under various (patho)physiological conditions, such as inflammation, obesity, or pregnancy. The phenotyping approach assumes that changes in plasma clearance of probe drugs are driven by changes in CYP enzyme activity. However, plasma clearance is also influenced by protein binding, blood-to-plasma ratio, and hepatic blood flow, all of which may change under (patho)physiological conditions. Using a physiologically based pharmacokinetic (PBPK) workflow, we aimed to evaluate whether the plasma clearance of commonly used CYP probe drugs is indeed directly proportional to alterations in CYP enzyme activity (sensitivity), and to what extent alterations in protein binding, blood-to-plasma ratio, and hepatic blood flow observed under (patho)physiological conditions impact plasma clearance (specificity). Plasma clearance of CYP probe drugs is sensitive to alterations in CYP enzyme activity, since alterations in intrinsic clearance between - 90% and + 150% resulted in near-proportional changes in plasma clearance, except for midazolam in the case of > 50% CYP3A4 induction. However, plasma clearance also changed near-proportionally with alterations in the unbound drug fraction, diminishing probe specificity. This was particularly relevant for high protein-bound probe drugs, as alterations in plasma protein binding resulted in larger relative changes in the unbound drug fraction. Alterations in the blood-to-plasma ratio and hepatic blood flow of ± 50% resulted in plasma clearance changes of less than ± 16%, meaning they limitedly impacted plasma clearance of CYP probe drugs, except for midazolam. In order to correct for the impact of non-metabolic determinants on probe drug plasma clearance, an R script was developed to calculate how much the CYP enzyme activity is actually altered under (patho)physiological conditions, when alterations in the unbound drug fraction, blood-to-plasma ratio, and/or hepatic blood flow also impact probe drug plasma clearance. As plasma protein binding can change under (patho)physiological conditions, alterations in unbound drug fraction should be accounted for when using CYP probe drug plasma clearance as a proxy for CYP enzyme activity in patient populations. The tool developed in this study can support researchers in determining alterations in CYP enzyme activity in patients with (patho)physiological conditions.

Keratoconus.

Keratoconus is a progressive eye disorder primarily affecting individuals in adolescence and early adulthood. The ectatic changes in the cornea cause thinning and cone-like steepening leading to irregular astigmatism and reduced vision. Keratoconus is a complex disorder with a multifaceted aetiology and pathogenesis, including genetic, environmental, biomechanical and cellular factors. Environmental factors, such as eye rubbing, UV light exposure and contact lens wearing, are associated with disease progression. On the cellular level, a complex interplay of hormonal changes, alterations in enzymatic activity that modify extracellular membrane stiffness, and changes in biochemical and biomechanical signalling pathways disrupt collagen cross-linking within the stroma, contributing to structural integrity loss and distortion of normal corneal anatomy. Clinically, keratoconus is diagnosed through clinical examination and corneal imaging. Advanced imaging platforms have improved the detection of keratoconus, facilitating early diagnosis and monitoring of disease progression. Treatment strategies for keratoconus are tailored to disease severity and progression. In early stages, vision correction with glasses or soft contact lenses may suffice. As the condition advances, rigid gas-permeable contact lenses or scleral lenses are prescribed. Corneal cross-linking has emerged as a pivotal treatment aimed at halting the progression of corneal ectasia. In patients with keratoconus with scarring or contact lens intolerance, surgical interventions are performed.

Влияние на климатичните промени върху хематологичния и биохимичен статус на стадо крави от порода Лимузин в средата на два последователни пасищни сезона

The aim of the present study was to evaluate the status in the red and white blood count, together with the values of some enzymes and metabolites - ALT, AST, ALP, serum iron and glucose in selected animals from a herd of Limousine beef cattle in the middle of two consecutive grazing seasons, with large differences in the average daily temperature, the amount and distribution of precipitation and of the quantity and quality of the grass at the end of its vegetation. The study was conducted in the experimental base “Zlatusha” of the IAS-Kostinbrod with 15 pregnant cows equalized by BCS and age, from which blood samples were taken in July, 2022 and 2023, by venipuncture of the tail vein. The analysis of hematological indicators in whole blood was performed with a 5-Part-Diff Automated Hematology Analyzer URIT-5160, and the analysis of biochemical indicators - in plasma extracted from it with a Semi-automatic Biochemical Analyzer BTS-350. Results are expressed as mean values ± S.E.M. and were analyzed statistically by the analysis of variance (ANOVA) method. Although there was almost no difference in the levels of hematocrit and the number of erythrocytes in the middle of two grazing seasons, in July, 2022, the studied cows had a significantly higher level of hemoglobin than in July, 2023 (P&lt;0.001), which reflected also on the significance of the difference in mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) – also at P&lt;0.001. There were no significant differences in leukocyte counts and leukocyte subpopulations and in iron and glucose concentrations in the middle of two consecutive years. ALT and AST enzyme activities were significantly lower (P&lt;0.05), and ALP activity was significantly higher in July, 2022 (P&lt;0.01).

Impact of Triclosan on Bacterial Biodiversity and Sediment Enzymes - A Microcosm Study.

Triclosan (TCS), a widely used antimicrobial biocide, has raised serious concern among the scientific community in recent years owing to its ubiquitous presence around the globe and toxicity to aquatic organisms. The current study investigated the alterations in bacterial diversity, nutrients, and sediment enzyme activity in TCS-exposed sediment. TCS concentrations of 3mg/L (T1) and 6mg/L (T2) were applied in a microcosm setup for 28 days to sediment collected from Versova Creek, Mumbai. Among sediment enzymes, dehydrogenase activity exhibited the greatest degree of variability in 3mg/L exposed sediment. Nitrite, total nitrogen and urease exhibited higher concentrations in 6mg/L TCS exposed sediment. The concentration of ammonia was observed to be decreasing in treatments exposed to 6mg/L TCS. Total heterotrophic bacteria exhibited an increase in count in T1 and a decrease in T2. Metagenomics data showed a higher relative abundance of bacteria in T1 compared to T2 on the 28th day of sampling. Proteobacteria was found to be the most abundant phylum in all samples, and their relative abundance was reduced by 0.14% in T1 and 5.48% in T2. The results confirm the alterations in the composition of sediment bacterial communities and their enzymatic activities due to TCS exposure.

A-134 Comparative Analysis of ALT and AST Measurements: Impact of P5P Activators on Platform Discrepancies

Abstract Background Vitamin B6 (VB6), notably its active form pyridoxal 5'-phosphate (P5P or PLP), is essential for ALT and AST enzyme activity in amino acid metabolism. Insufficient P5P can yield inaccurately low ALT and AST measurements in individuals with VB6 deficiency. This study assesses VB6 deficiency prevalence and its impact on ALT and AST measurements on Beckman Coulter compared to Roche and Siemens platforms. Methods VB6 levels were analyzed in 996 patients, with &amp;lt; 20 nmol/L considered deficient. ALT and AST levels were compared between Beckman Coulter AU5800 versus Roche cobas c, and Beckman Coulter AU5800 versus Siemens Dimension Vista. Roche and Siemens assays included P5P supplements, while Beckman Coulter did not. 29 ALT specimens (23 VB6 deficient and 6 normal) and 34 AST specimens (28 VB6 deficient and 6 normal) were compared between Beckman Coulter and Roche. Additionally, 16 specimens (4 VB6 deficient and 12 normal) were tested for ALT and AST for the comparison between Beckman Coulter and Siemens. The study then assessed the impact of P5P-containing reagents on test results by comparing the percentage differences in average concentration and their clinical decision implications across different platforms, within the low, normal, and high ranges. Results Out of 996 patients, 356 exhibited VB6 results below 20 nmol/L, indicating a striking 36% prevalence of VB6 deficiency within our patient population. Beckman Coulter and Roche comparison: In VB6 deficient patients, ALT displayed an average concentration difference of 11.5%: Beckman ALT mean 23 (+/-24.6) U/L versus Roche ALT mean 26 (+/-30) U/L. AST demonstrated a 41% difference in average concentration: Beckman AST mean 28.2 (+/-28.9) U/L versus Roche AST 48.9 (+/-49) U/L. In normal patients, ALT displayed an average concentration difference of 2.4%: Beckman ALT mean 21.3 (+/-21.3) U/L versus Roche ALT mean 20.8 (+/-25.7) U/L. AST demonstrated a 23.5% difference in average concentration: Beckman AST mean 25.5. (+/-25.4) U/L versus Roche AST 33.3 (+/-37.5) U/L. Additionally, 13% of ALT and 18% of AST displayed noteworthy effects on clinical decisions, resulting in shifts from low abnormal to normal range or normal range to high abnormal. Beckman Coulter and Siemens comparison: In VB6 deficient patients, ALT displayed an average concentration difference of 36.8%: Beckman ALT mean 18 (+/-15.7) U/L versus Siemens ALT mean 28.5 (+/-20.5) U/L. AST demonstrated a 43.4% difference in average concentration: Beckman AST mean 18.3 (+/-8.1) U/L versus Siemens AST 32.3 (+/-17.2) U/L. In normal patients, ALT displayed an average concentration difference of 32.8%: Beckman ALT mean 22.2 (+/-16.1) U/L versus Siemens ALT mean 33.1 (+/-20.1) U/L. AST demonstrated a 31.3% difference in average concentration: Beckman AST mean 25.4 (+/-12.3) U/L versus Siemens AST 36.1 (+/-19.7) U/L. While 6% of ALT specimens would have altered clinical decisions, the impact was more pronounced for AST, with 50% of results changing to an upper reference point. Conclusions In clinical settings where accurate assessment of enzyme activity is vital for effective patient care, our study emphasizes the crucial necessity for standardizing AST and ALT methodologies to incorporate P5P supplementation.

CYP P450 and non-CYP P450 Drug Metabolizing Enzyme Families Exhibit Differential Sensitivities towards Proinflammatory Cytokine Modulation.

Compromised hepatic drug metabolism in response to proinflammatory cytokine release is primarily attributed to downregulation of cytochrome P450 (CYP) enzymes. However, whether inflammation also affects other phase I and phase II drug metabolizing enzymes (DMEs), such as the flavin monooxygenases (FMOs), carboxylesterases (CESs), and UDP glucuronosyltransferases (UGTs), remains unclear. This study aimed to decipher the impact of physiologically relevant concentrations of proinflammatory cytokines on expression and activity of phase I and phase II enzymes, to establish a hierarchy of their sensitivity as compared with the CYPs. Hereto, HepaRG cells were exposed to interleukin-6 and interleukin-1β to measure alterations in DME gene expression (24 h) and activity (72 h). Sensitivity of DMEs toward proinflammatory cytokines was evaluated by determining IC50 (potency) and Imax (maximal inhibition) values from the concentration-response curves. Proinflammatory cytokine treatment led to nearly complete downregulation of CYP3A4 (∼98%) but was generally less efficacious at reducing gene expression of the non-CYP DME families. Importantly, FMO, CES, and UGT family members were less sensitive toward interleukin-6 induced inhibition in terms of potency, with IC50 values that were 4.3- to 7.4-fold higher than CYP3A4. Similarly, 18- to 31-fold more interleukin-1β was required to achieve 50% of the maximal downregulation of FMO3, FMO4, CES1, UGT2B4, and UGT2B7 expression. The differential sensitivity persisted at enzyme activity level, highlighting that alterations in DME gene expression during inflammation are predictive for subsequent alterations in enzyme activity. In conclusion, this study has shown that FMOs, CESs, and UGTs enzymes are less impacted by IL-6 and IL-1β treatment as compared with CYP enzymes. SIGNIFICANCE STATEMENT: While the impact of proinflammatory cytokines on CYP expression is well established, their effects on non-CYP phase I and phase II drug metabolism remains underexplored, particularly regarding alterations in drug metabolizing enzyme (DME) activity. This study provides a quantitative understanding of the sensitivity differences to inflammation between DME family members, suggesting that non-CYP DMEs may become more important for the metabolism of drugs during inflammatory conditions due to their lower sensitivity as compared with the CYPs.

Low temperature and high humidity affect dynamics of chlorophyll biosynthesis and secondary metabolites in Cucumber.

During the cold season, low temperature (LT) and high relative humidity (HRH) are significant environmental factors in greenhouses and plastic tunnels, often hindering plant growth and development. The chlorophyll (Chl) biosynthesis inhibitory mechanisms under LT and HRH stress are still widely unclear. To understand how cucumbers seedlings respond to LT and HRH stress, we investigated the impact of these stressors on Chl biosynthesis. Our results revealed that individual LT, HRH and combined LT + HRH stress conditions affected chlorophyll a, b, total chlorophyll and carotenoid content, reducing the levels of these pigments. The levels of Chlorophyll precursors were also markedly reduced under LT and HRH stresses, with the greatest reduction observed in cucumber seedlings exposed to LT + HRH conditions (9/5℃, 95%HRH). The activities of glutamate-1-semialdehyde transaminase (GSA-AT), ALA dehydratase (ALAD), Mg-chelatase, and protochlorophyllide oxidoreductase (POR) were increased under individual LT, HRH, conditions but decreased by combination of LT + HRH stress condition. In addition, Chl biosynthesis related genes (except PBG) were upregulated by the HRH stress but were significantly downregulated under the LT + HRH stress condition in cucumber seedlings. Furthermore, the content of phenols, flavonoids and phenolic acids (cinnamic acid and caffeic acid) were significantly surged under LT + HRH treatment over the control. Histochemical observation showed higher O2- and H2O2 content in cucumber leaves during the LT and HRH stress. The results indicate that LT + HRH stress significantly impairs chlorophyll biosynthesis in cucumber seedlings by drastically reducing pigment accumulation, altering enzyme activity and gene expression. Additionally, LT + HRH stress induces oxidative damage, which further exacerbates the decline in chlorophyll content and affects overall cucumber metabolism.

A Review of Electromagnetic Fields in Cellular Interactions and Cacao Bean Fermentation.

The influence of magnetic fields on biological systems, including fermentation processes and cocoa bean fermentation, is an area of study that is under development. Mechanisms, such as magnetosensitivity, protein conformational changes, changes to cellular biophysical properties, ROS production, regulation of gene expression, and epigenetic modifications, have been identified to explain how magnetic fields affect microorganisms and cellular processes. These mechanisms can alter enzyme activity, protein stability, cell signaling, intercellular communication, and oxidative stress. In cacao fermentation, electromagnetic fields offer a potential means to enhance the sensory attributes of chocolate by modulating microbial metabolism and optimizing flavor and aroma development. This area of study offers possibilities for innovation and the creation of premium food products. In this review, these aspects will be explored systematically and illustratively.

Prothioconazole Stress Reduces Bacterial Richness and Alters Enzyme Activity in Soybean Rhizosphere.

Prothioconazole (PTC) is currently a popular triazole fungicide. In recent years, as the use of PTC has increased, there has been growing concern about its environmental and toxicological effects. Here, we studied the effect of PTC on the growth of soybean plants and further analyzed the enzyme activity and microbial community of rhizosphere soil after PTC treatment through 16S rRNA gene high-throughput sequencing and fungal ITS. Changes in structural diversity and species richness were measured using Simpson's diversity index, Shannon's diversity index and the Chao1 and ACE algorithms. The statistical t-test was applied to test whether the index values were significantly different between the two groups. The results showed that the contents of malondialdehyde (MDA) and H2O2 increased after the recommended dose of PTC, indicating that PTC has a strong toxic effect on plant growth, thus affecting the healthy growth of plants. In the presence of PTC, the species richness of fungi and bacteria decreased in all three soil types (black soil, yellow earth and red earth), and the community structure also changed significantly (the p-values were all less than 0.05). Proteobacteria, Actinomycetota, Bacteroidota and Acidobacteriota were the main bacteria, and the abundance of Acidobacteriota and Chloroflexi increased. The dominant fungal communities were Ascomycota and Mortierellomycota. The increased abundance of potentially beneficial microorganisms, such as Sphingomonadaceae, suggested that plants may be resistant to PTC stress by recruiting beneficial microorganisms. PICRUSt analysis showed that the metabolism-related functions and membrane transport pathway of rhizosphere bacterial community were inhibited after PTC stress. Spearman correlation analysis revealed a weak correlation between key fungal taxa and rhizosphere variables in the presence of PTC. Therefore, compared with those in the fungal community, the bacterial community was more likely to help plants resist PTC stress, indicating that these key fungal groups may indirectly help soybean growth under PTC stress by affecting the bacterial community.

Assessing the Effects of a Diet of BPA Analogue-Exposed Microalgae in the Clam Ruditapes philippinarum.

In our previous study, we demonstrated that the microalgae Phaeodactylum tricornutum can bioaccumulate bisphenol A analogues. Since this microalgae species is part of the diet of marine filter-feeding organisms, such as bivalves, in this study we tested the hypothesis that a diet based on exposed microalgae can exert negative effects on the clam Ruditapes philippinarum. Microalgae were exposed for 7 days to 300 ng/L of bisphenol AF (BPAF), bisphenol F (BPF), and bisphenol S (BPS), alone or as a mixture (MIX), to allow bioaccumulation. Microalgae were then supplied as food to bivalves. After 7 and 14 days of diet, the effects of exposed microalgae were evaluated on a battery of biomarkers measured in haemolymph/haemocytes, gills and digestive glands of clams. In addition, bioaccumulation of the three bisphenols was investigated in clams by UHPLC-HRMS. The results obtained demonstrated that total haemocyte count (THC) increased in clams following ingestion for 7 days of BPAF- and BPF-exposed microalgae, while BPS-exposed microalgae significantly reduced THC after 14 days of diet. MIX- and BPS-exposed microalgae increased haemocyte proliferation. The diet of exposed microalgae affected acid and alkaline phosphatase activity in clams, with an opposite response between haemolymph and haemocytes. Regarding antioxidants, an increase in catalase activity was observed in clams after ingestion of BPA analogue-exposed microalgae. The results also demonstrated marked oxidative stress in gills, the first tissue playing an important role in the feeding process. Oxidative damage was recorded in both the gills and digestive glands of clams fed BPA analogue-exposed microalgae. Alterations in epigenetic-involved enzyme activity were also found, demonstrating for the first time that BPA analogue-exposed food can alter epigenetic mechanisms in marine invertebrates. No bioaccumulation of BPA analogues was detected in clam soft tissues. Overall, this study demonstrated that a diet of BPA analogue-exposed microalgae can induce significant alterations of some important biological responses of R. philippinarum. To our knowledge, this is the first study demonstrating the effects of ingestion of BPA analogue-exposed microalgae in the clam R. philippinarum, suggesting a potential ecotoxicological risk for the marine food chain, at least at the first levels.

Adaptability to the environment of protease by secondary structure changes and application to enzyme-selective hydrolysis

The reactions involving enzymes are significantly influenced by various environmental factors. Clarity of how the activity and structure of proteases impact their function is crucial for more efficient application of enzymes as a tool. The impact of temperature, pH, and ionic strength on changes in protease activity, secondary structure, and protein conformation during enzymatic hydrolysis were investigated in this study. The enzymatic activity and secondary structure of acid-base protease were found to undergo significant modifications under different physical conditions, as demonstrated by UV spectrophotometry and FTIR spectroscopy analysis. Specifically, variations in α-helix and β-fold content were observed to correlate with changes in enzyme activity. Molecular simulation analysis revealed that physical conditions have varying effects on the protease, particularly influencing enzyme activity and secondary structure. Evaluation of the proteases indicated alterations in both enzyme activity and structure. This treatment selectively hydrolyzed β-lactoglobulin and reduced sensitization. These findings offer novel perspectives on the functionalities and regulatory mechanisms of proteases, as well as potential industrial applications.

The gradual establishment of complex coumarin biosynthetic pathway in Apiaceae

Complex coumarins (CCs) represent characteristic metabolites found in Apiaceae plants, possessing significant medical value. Their essential functional role is likely as protectants against pathogens and regulators responding to environmental stimuli. Utilizing genomes and transcriptomes from 34 Apiaceae plants, including our recently sequenced Peucedanum praeruptorum, we conduct comprehensive phylogenetic analyses to reconstruct the detailed evolutionary process of the CC biosynthetic pathway in Apiaceae. Our results show that three key enzymes – p-coumaroyl CoA 2’-hydroxylase (C2’H), C-prenyltransferase (C-PT), and cyclase – originated successively at different evolutionary nodes within Apiaceae through various means of gene duplications: ectopic and tandem duplications. Neofunctionalization endows these enzymes with novel functions necessary for CC biosynthesis, thus completing the pathway. Candidate genes are cloned for heterologous expression and subjected to in vitro enzymatic assays to test our hypothesis regarding the origins of the key enzymes, and the results precisely validate our evolutionary inferences. Among the three enzymes, C-PTs are likely the primary determinant of the structural diversity of CCs (linear/angular), due to divergent activities evolved to target different positions (C-6 or C-8) of umbelliferone. A key amino acid variation (Ala161/Thr161) is identified and proven to play a crucial role in the alteration of enzymatic activity, possibly resulting in distinct binding forms between enzymes and substrates, thereby leading to different products. In conclusion, this study provides a detailed trajectory for the establishment and evolution of the CC biosynthetic pathway in Apiaceae. It explains why only a portion, not all, of Apiaceae plants can produce CCs and reveals the mechanisms of CC structural diversity among different Apiaceae plants.

Mitigating ethyl carbamate in Chinese rice wine: Role of raspberry extract

This study investigated the use of raspberry extract (RBE) in mitigating ethyl carbamate (EC) accumulation in Chinese rice wine (Huangjiu), a traditional fermented beverage. It focused on the addition of RBE to the fermentation mash and its effects on EC levels. Results indicated a significant reduction in EC production, attributed to RBE's role in altering urea and citrulline catabolism and inhibiting arginine metabolism, thus preventing EC precursors from reacting with ethanol. Additionally, RBE enhanced the rice wine's flavor profile, as shown by volatile component and amino acid analysis. This study also explored RBE's impact on Saccharomyces cerevisiae (S. cerevisiae) arginine metabolism in a simulated fermentation environment, observing increased arginine, reduced urea and citrulline levels, altered enzyme activities, and gene expression changes in arginine metabolism and transport pathways. In conclusion, it clearly demonstrated RBE's efficacy in reducing EC content in Chinese rice wine, offering valuable insights for EC reduction strategies.

Assessment of waterlogging-induced changes in enzymatic antioxidants and carbohydrate metabolism in peanuts genotypes

Soil flooding, manifesting as submergence or waterlogging stress, significantly impacts plant species composition and agricultural productivity, particularly in regions with low rainfall. This study investigates the biochemical responses of two peanut (Arachis hypogaea L.) genotypes, DH-86 and GJG-32, under waterlogging stress. The experiment involved in-vivo pot trials where peanut plants were subjected to continuous waterlogging for 12 days at the flowering stage. Biochemical analyses of leaves conducted and revealed significant alterations in enzyme activities and metabolite concentrations. Key findings include variations in superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPOD), α-amylase, invertase, acid phosphomonoesterase activities, and changes in starch, proline, reducing sugars, and chlorophyll content. SOD, CAT, and GPOD activities exhibited differential responses between genotypes, highlighting DH-86's quicker recovery post-waterlogging. Notably, DH-86 demonstrated higher resilience, reflected in its rapid normalization of biochemical parameters, while GJG-32 showed prolonged stress effects. These findings underscore the importance of antioxidative enzyme systems in mitigating oxidative damage induced by waterlogging. This study enhances our understanding of the biochemical adaptations of peanut genotypes to waterlogging stress, offering valuable insights for breeding programs focused on improving flood tolerance in crops.

Enzyme-Triggered Degradation of Supramolecularly Cross-Linked Polymersomes of Azobenzene-Based Polyurethane: Cell-Selective Anticancer Drug Release.

Enzyme-responsive self-assembled nanostructures for drug delivery applications have gained a lot of attention, as enzymes exhibit dysregulation in many disease-associated microenvironments. Azoreductase enzyme levels are strongly elevated in many tumor tissues; hence, here, we exploited the altered enzyme activity of the azoreductase enzyme and designed a main-chain azobenzene-based amphiphilic polyurethane, which self-assembles into a vesicular nanostructure and is programmed to disassemble in response to a specific enzyme, azoreductase, with the help of the nicotinamide adenine dinucleotide phosphate (NADPH) coenzyme in the hypoxic environment of solid tumors. The vesicular nanostructure sequesters, stabilizes the hydrophobic anticancer drug, and releases the drug in a controlled fashion in response to enzyme-triggered degradation of azo-bonds and disruption of vesicular assembly. The biological evaluation revealed tumor extracellular matrix pH-induced surface charge modulation, selective activated cellular uptake to azoreductase overexpressed lung cancer cells (A549), and the release of the anticancer drug followed by cell death. In contrast, the benign nature of the drug-loaded vesicular nanostructure toward normal cells (H9c2) suggested excellent cell specificity. We envision that the main-chain azobenzene-based polyurethane discussed in this manuscript could be considered as a possible selective chemotherapeutic cargo against the azoreductase overexpressed cancer cells while shielding the normal cells from off-target toxicity.