Enzyme Activity Assays Research Articles

Antibacterial mechanism of atmospheric cold plasma against Pseudomonas fluorescens and Pseudomonas putida and its preservation application on in-packaged red shrimp paste

This study aimed to investigate the antibacterial mechanism of atmospheric cold plasma (ACP) against Pseudomonas fluorescens and Pseudomonas putida and its preservation effect on red shrimp paste. A reactive species (RS) assay showed that O3, H2O2, and total nitric oxide were generated after ACP treatment, which possessed great potential for antibacterial and food preservation. In vitro antibacterial results showed that excess RS inhibited bacterial activity through cell membrane damage. Molecular docking predictions and enzyme activity assays indicated that ACP-induced RS might deactivate dehydrogenases (such as malic dehydrogenase) by oxidatively modifying the active sites. Fluorescence quantification experiments validated the damage of RS to dsDNA. Further preservation tests on shrimp paste demonstrated that ACP treatment significantly delayed the increase in total viable count, Pseudomonas count, and total volatile basic‑nitrogen during refrigeration. This study deepened the understanding of the antibacterial mechanism of ACP and highlighted its potential application as a new preservation method.

Comparative study of immune responses and intestinal microbiota in the gut-liver axis between wild and farmed pike perch (Sander Lucioperca).

Pike perch (Sander Lucioperca) is a predatory freshwater fish, which is highly popular amongst consumers, owing to its white flesh with a delicate structure and mild flavor. Compared to wild pike perch, the diet of farmed ones has shifted from natural food to artificial feeds. These changes would affect the gut flora of the pike perch. Endogenous metabolites of the intestinal flora are transferred through the gut-liver axis, which affects the physiological functions of the host. By studying wild and farmed individuals of the pike perch, novel insights into the stability of the intestinal flora can be provided. In this study, we measured various immune parameters in the blood, liver and intestine of wild and farmed pike perch using enzyme activity assays and real-time fluorescence quantitative PCR. Gut microbes were also collected for 16S rRNA gene sequencing. Our results showed that the serum low-density lipoprotein cholesterol (LDL-C) levels were twice as high in the wild group as in the farmed group. Furthermore, the activities of glutamate pyruvate transaminase (GPT) and glutamate oxaloacetate transaminase (GOT) in the intestinal tissues of the wild group were 733.91 U/g and 375.35 U/g, which were significantly higher than those of the farmed group. Expression of IL10 in the liver of farmed pike perch was also 4-fold higher than that of wild pike perch. The expression of genes related to the p53-BAX/Bcl2 signaling pathway was higher in both intestinal and liver tissues of wild pike perch compared with farmed. 16S rRNA gene analysis of the gut microflora showed a high relative abundance of Cetobacterium in the gut of farmed pike perch. As a result, our study indicates that dietary differences affect the diversity, composition and relative abundance of the gut flora of the pike perch. Meanwhile, it affects the glycolipid metabolism and immunomodulation of pike perch.

Full-Length Transcriptome Analysis and Characterization of DFRs Involved in the Formation of Anthocyanin in Allium wallichii

Allium wallichii is famous for its reddish-purple flowers, which can be utilized as cut flowers and garden landscaping. Flower color is mainly determined by flavonoids, betalains, carotenoids, as well as other pigments. However, there is no research on the color formation mechanism in A. wallichii, which restricts its genetic improvement and development of superior varieties. The flower of A. wallichii was collected for full-length transcriptome sequencing and metabolome analysis using PacBio SMART and UPLC-MS, respectively. A total of 45 anthocyanins were detected in its flower, and 75,778 transcripts of 107,208 non-redundant transcripts were annotated. Then, two AwDFRs were cloned and characterized using bioinformatics tools. Enzyme activity assays revealed that both AwDFR1 and AwDFR2 possessed DFR activity in vitro that only accepted DHQ and DHM as substrates, except for DHK. Finally, physiological results showed that AwDFR1 and AwDFR2 could restore the lacking phenotypes of Arabidopsis tt3 mutant and increase the content of anthoycanin in tobacco petals. The anthocyanins and transcriptome in A. wallichii were firstly reported, and AwDFR1 and AwDFR2 are key enzymes participating in the biosynthesis of anthocyanins. This research provides important guidance for future key gene mining, color improvement, and horticultural breeding in A. wallichii.

7963 Analysis of the inhibition of adrenal synthesis and peripheral metabolism of corticosteroids by oteseconazole and two preclinical tetrazole antifungals

Abstract Disclosure: M. Jäger: None. V. González-Ruiz: None. F.L. Joos: None. D.V. Winter: None. J. Boccard: None. T. Degenhardt: Employee; Self; Mycovia Inc. S. Brand: Employee; Self; Mycovia Inc.. S. Rudaz: None. G.R. Thompson: None. A. Odermatt: None. The triazole antifungals posaconazole and itraconazole can cause pseudohyperaldosteronism with hypertension and hypokalemia by blocking adrenal cortisol synthesis and its peripheral inactivation [1]. Mechanisms of pseudohyperaldosteronism include inhibition of adrenal cytochrome P450 enzymes CYP11B1 and CYP17A1, as well as peripherally expressed 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2). To enhance specificity for fungal CYP51, tetrazole compounds have been developed [2]. Here, we used adrenal H295R cells and enzyme activity assays to assess the inhibition of steroidogenesis by the tetrazoles oteseconazole, VT-1129 and VT-1598. In addition, the three tetrazoles were tested for inhibition of 11β-HSD2. Steroidomic footprint analyses of H295R cell supernatants using untargeted LC-HRMS with steroid annotation indicated common features of the oteseconazole and VT-1129 patterns with that of the triazole itraconazole, and similarities between VT-1598 and the triazole isavuconazole. Targeted quantification of nine adrenal steroids by UHPLC-MS/MS in supernatants of treated H295R cells showed an overall inhibition of steroidogenesis by the tetrazoles and the triazoles itraconazole and isavuconazole. Experiments using recombinant human enzymes suggested that this effect is not caused by direct inhibition of steroidogenic enzymes because no or very weak inhibition could be observed. Furthermore, oteseconazole, VT-1129 and VT-1598 did not inhibit 11β-HSD2, suggesting that they unlikely cause pseudohyperaldosteronism. Further research needs to uncover the exact mechanism for the observed overall inhibition of steroidogenesis by the tetrazoles as well as itraconazole and isavuconazole, and whether concentrations reached in patients are high enough to cause adrenal insufficiency and hyperplasia.

New insight into Clostridium butyricum-ferroferric oxide hybrid system in exogenous carbon dioxide-assisted anaerobic fermentation for acetate and butyrate production

Mixotrophic cultivation, utilizing both gas and organic substances, is commonly employed to minimize the carbon loss during anaerobic fermentation of bulk chemicals. Herein, a novel Clostridium butyricum-ferroferric oxide (Fe3O4) hybrid system, enhanced by exogenous carbon dioxide (CO2), was proposed to improve carbon recovery and optimize metabolite production. The results demonstrated that exogenous CO2 improved metabolite selectivity towards acetate/butyrate, while also accelerating CO2 fixation. Compared to pure Clostridium butyricum, the hybrid system significantly increased carbon conversion to primary metabolites, boosting butyrate and acetate production by 18.7 % and 18.4 %, respectively. Enzyme activity assays revealed that Fe3O4 and exogenous CO2 acted synergistically, enhancing the activities of key enzymes involved in CO2 assimilation. Additionally, Fe3O4 facilitated intra- and extracellular electron transfer, further improving the fermentation process. This study offers new insights into the combined effects of exogenous CO2 and Fe3O4 on anaerobic fermentation, providing an efficient strategy for carbon recovery and selective acetate/butyrate production.

Bifenthrin at Sublethal Concentrations Suppresses Mating and Laying of Female Conogethes punctiferalis by Regulating Sex Pheromone Biosynthesis and JH Signals.

Conogethes punctiferalis, a polyphagous pest in Asia, infests various crops, causing severe economic losses. Its larvae feed inside plants, making management challenging, with conventional insecticides. This study examines sublethal bifenthrin effects on the reproductive capabilities of adult females. Findings show sublethal bifenthrin concentrations (LC1, LC10, LC20, and LC30) significantly reduce sex pheromone production and mating success in a dose-dependent manner. Furthermore, these sublethal exposures influence the expression of pheromone biosynthesis activating neuropeptide and key juvenile hormone signaling genes, including methoprene-tolerant and Krüppel-homologue 1. Enzyme activity assays and metabolite measurements indicated that sublethal bifenthrin exposure decreases trehalose and pyruvic acid levels, suppressing the enzyme activities required for sex pheromone biosynthesis. Additionally, bifenthrin exposure delays ovarian development, reduces ovary size, and decreases egg production and hatchability. These results suggest bifenthrin's potential in attract-and-kill strategies by disrupting essential pathways for pest control, offering insights for improved insecticide use and innovative pest management for C. punctiferalis.

Identification of a 301 bp promoter core region of the SrUGT91D2 gene from Stevia rebaudiana that contributes to hormone and abiotic stress inducibility

BackgroundThe UDP-glucuronosyltransferase 91D2 (SrUGT91D2) gene is a crucial element in the biosynthetic pathway of steviol glycosides (SGs) and is responsible for creating 1,2-β-D glucosidic bonds at the C19 and C13 positions. This process plays a vital role in the synthesis of rebaudioside M (RM) and rebaudioside D (RD). The promoter, which regulates gene expression, requires functional analysis to understand gene expression regulation. However, investigations into the function of the promoter of SrUGT91D2 (pSrUGT91D2) have not been reported.ResultsThe pSrUGT91D2 was isolated from six S. rebaudiana lines, and subsequent multiple sequence comparisons revealed the presence of a 26 bp inDel fragment (pSrUGT91D2-B1188 type) in lines GP, GX, 110, 1114, and B1188 but not in the pSrUGT91D2 of line 023 (pSrUGT91D2-023 type). Bioinformatics analysis revealed a prevalence of significant cis-regulatory elements (CREs) within the promoter sequences, including those responsive to abscisic acid, light, anaerobic conditions, auxin, drought, low temperature, and MeJA. To verify the activity of pSrUGT91D2, the full-length promoter and a series of 5’ deletion fragments (P1–P7) and a 3’ deletion fragment (P8) from various lines were fused with the reporter β-glucuronidase (GUS) gene to construct the plant expression vector, pCAMBIA1300-pro∷GUS. The transcriptional activity of these genes was examined in tobacco leaves through transient transformation. GUS tissue staining analysis and enzyme activity assays demonstrated that both the full-length promoter and truncated pSrUGT91D2 were capable of initiating GUS expression in tobacco leaves. Interestingly, P8-pSrUGT91D2-B1188 (containing the inDel segment, 301 bp) exhibited enhanced activity in driving GUS gene expression. Transient expression studies of P8-pSrUGT91D2-B1188 and P8-pSrUGT91D2-023 in response to exogenous hormones (abscisic acid and indole-3-acetic acid) and light indicated the necessity of the inDel region for P8 to exhibit transcriptional activity, as it displayed strong responsiveness to abscisic acid (ABA), indole-3-acetic acid (IAA), and light induction.ConclusionsThese findings contribute to a deeper understanding of the regulatory mechanism of the upstream region of the SrUGT91D2 gene and provide a theoretical basis for future studies on the interaction between CREs of pSrUGT91D2 and related transcription factors.

Baseline sensitivity and resistance risk of Sclerotinia sclerotiorum to glabridin and the possible anti-fungal mechanism

Sclerotinia stem rot caused by Sclerotinia sclerotiorum is one of the most serious diseases of oilseed rape. Chemical control is an important method to control this disease, however, development of fungal resistance to commonly used fungicides has led to severe yield losses in recent years. Therefore, development of novel fungicides against S. sclerotiorum is urgently needed. Glabridin is one of the major flavonoids in Glycyrrhiza L. plants, and we previously found that it is very effective against S. sclerotiorum. Nevertheless, the baseline sensitivity and resistance risk of S. sclerotiorum to glabridin as well as the possible anti-fungal mechanism need further elucidation. In this study, we revealed that the EC50 (median effective concentration) values of glabridin against 109 S. sclerotiorum isolates collected from Jiangsu Province of China ranged from 0.51 to 8.03 μg/mL with a mean EC50 value of 3.05 ± 1.27 μg/mL. No cross-resistance was observed between glabridin and carbendazim, and no glabridin-resistant mutants were obtained by chemical induction. RNA profiling result showed that tyrosine metabolism of S. sclerotiorum were evidently affected by glabridin. qRT-PCR, enzyme activity assay, and molecular docking proved that glabridin greatly reduced both the expression level and enzyme activity of tyrosinase in S. sclerotiorum. Furthermore, S. sclerotiorum incurred certain impairment in its membrane integrity after glabridin treatment at 10 μg/mL. This study is the first report on baseline sensitivity and resistance risk of S. sclerotiorum to glabridin, and it is revealed that glabridin may interfere tyrosine metabolism and membrane integrity of S. sclerotiorum.

First fragment-based screening identifies new chemotypes inhibiting ERAP1-metalloprotease

Inhibition of endoplasmic reticulum aminopeptidase 1 (ERAP1) by small-molecules is being eagerly investigated for the treatment of various autoimmune diseases and in the field of immuno-oncology after its active involvement in antigen presentation and processing. Currently, ERAP1 inhibitors are at different stages of clinical development, which highlights its significance as a promising drug target. In the present work, we describe the first-ever successful identification of several ERAP1 inhibitors derived from a fragment-based screening approach. We applied an enzymatic activity assay to a large library of ∼3000 fragment entries in order to retrieve 32 hits. After a multi-faceted selection process, we prioritized 3 chemotypes for SAR optimization and strategic modifications provided 2 series (2-thienylacetic acid and rhodanine scaffolds) with improved analogues at the low micromolar range of ERAP1 inhibition. We report also evidence of selectivity against homologous aminopeptidase IRAP, combined with complementary in silico docking studies to predict the binding mode and site of inhibition. Our compounds can be the starting point for future fragment growing and rational drug development, incorporating new chemical modalities.

Nobiletin, as a Novel PDE4B Inhibitor, Alleviates Asthma Symptoms by Activating the cAMP-PKA-CREB Signaling Pathway.

Asthma is a chronic airway inflammation that is considered a serious public health concern worldwide. Nobiletin (5,6,7,8,3',4'-hexamethyl flavonoid), an important compound isolated from several traditional Chinese medicines, especially Citri Reticulatae Pericarpium, is widely used for a number of indications, including cancer, allergic diseases, and chronic inflammation. However, the mechanism by which nobiletin exerts its anti-asthmatic effect remains unclear. In this research, we comprehensively demonstrated the anti-asthmatic effects of nobiletin in an animal model of asthma. It was found that nobiletin significantly reduced the levels of inflammatory cells and cytokines in mice and alleviated airway hyperresponsiveness. To explore the target of nobiletin, we identified PDE4B as the target of nobiletin through pharmacophore modeling, molecular docking, molecular dynamics simulation, SPR, and enzyme activity assays. Subsequently, it was found that nobiletin could activate the cAMP-PKA-CREB signaling pathway downstream of PDE4B in mouse lung tissues. Additionally, we studied the anti-inflammatory and anti-airway remodeling effects of nobiletin in LPS-induced RAW264.7 cells and TGF-β1-induced ASM cells, confirming the activation of the cAMP-PKA-CREB signaling pathway by nobiletin. Further validation in PDE4B-deficient RAW264.7 cells confirmed that the increase in cAMP levels induced by nobiletin depended on the inhibition of PDE4B. In conclusion, nobiletin exerts anti-asthmatic activity by targeting PDE4B and activating the cAMP-PKA-CREB signaling pathway.

Mechanistic insights into Suanzaoren Decoction's improvement of cardiac contractile function in anxiety-induced cardiac insufficiency

Ethnopharmacological relevanceAccording to traditional Chinese medicine, Anxiety-induced cardiac blood insufficiency leads to palpitations and restlessness. Suanzaoren Decoction (SD) is effective in replenishing blood and promoting blood circulation. Clinical practice has shown that it has a better therapeutic effect on cardiac insufficiency. However, its mechanism of action is still unclear. Aim of the studyThe study aims to determine the mechanism by which SD treats chronic restraint stress (CRS)-induced anxiety-induced cardiac insufficiency (ACI). Materials and methodsSD was orally administered to mice with CRS-induced ACI. Firstly, we constructed an anxiety model in mice by CRS. Subsequently, SD was investigated to assess cardiac function and pathological changes through echocardiography, H&E staining, and Masson staining. Thirdly, the function of sympathetic and parasympathetic nerves was evaluated using enzyme-linked immunosorbent assay (ELISA) and enzyme activity assays. Network pharmacology and molecular docking were employed to predict potential targets for SD treatment of cardiac insufficiency. CaMKII expression was scrutinized utilizing publicly accessible databases. CaMKII was identified as a target through immunohistochemistry and Western Blot analysis in mouse hearts. Finally, the therapeutic mechanism of SD was confirmed in injured cardiomyocytes via Western Blot and quantitative PCR. ResultsSD exerted anxiolytic effects by increasing the frequency of entries into and the duration spent in open arms while reducing the time spent in the light chamber and increasing the number of transitions between light and dark chambers. Additionally, it mitigated cardiac insufficiency, as evidenced by the enhancement of left ventricular ejection fraction (LVEF) and attenuation of cardiomyocyte damage and inflammatory infiltration.However, SD did not alleviate the elevated norepinephrine (NE) and decreased Acetylcholine (Ach) in anxiety states. To investigate the mechanism of action of SD, we constructed a Drug-Component-Target-Disease network, identifying 13 potential active compounds. Additionally, leveraging bioinformatics analysis and molecular docking targeting heart diseases characterized by clinical left ventricular ejection fraction (LVEF), we focused on the CaMKII target. The ability of SD to modulate CaMKII expression and phosphorylation in the mouse heart was investigated using immunohistochemistry and Western blotting. SD was found to alleviate NE-injured cardiomyocytes by modulating the Ca2+/CaMKII/MEF2 and GATA4 pathways. ConclusionSD is a potential formula for the treatment of chronic restraint stress (CRS)-induced ACI that ameliorates cardiomyocyte injury and improves cardiac function. Its efficacy is associated with the inhibition of the Ca2+/CaMKII/MEF2 and GATA4 signaling pathways.

Synthesis and Antifungal Activities of Glucosamine Aromatic Derivatives Against Four Phytopathogenic Fungi of Crops.

A series of diversified glucosamine derivatives (3a-3y) was synthesized and their antifungal activity was examined against four kinds of phytopathogens, Fusarium graminearum (F. graminearum), Fusarium moniliforme (F. moniliforme), Curvularia. lunata (C. lunata), and Rhizoctonia solani (R. solani) which cause seriously economic losses worldwide by affecting crops. The compound 3o showed remarkable antifungal activity against F. graminearum with EC50 value of 3.96 μg/mL, compared to the standard drug triadimefon (10.1 μg/mL). 3D-QSAR model with the statistically recommended values (r2=0.915, q2=0.872) showed that positive charge group or bulky group in the benzyl ring was favorable for the antifungal activity. Enzyme activity assays confirmed that 3o had a moderate inhibition of trehalase with inhibition rate of 51.4 % at 5 μg/mL, which is comparable to those of commercial inhibitor validamycin A with inhibition rate of 83.3 %. Molecular docking analysis revealed that 3o also had a hydrogen bond interaction with key amino acid residue compared to validoxylamine.

MecE, MecB, and MecC proteins orchestrate methyl group transfer during dichloromethane fermentation.

Dichloromethane (DCM), a common hazardous industrial chemical, is anaerobically metabolized by four bacterial genera: Dehalobacter, Dehalobacterium, Ca. Dichloromethanomonas, and Ca. Formimonas. However, the pivotal methyltransferases responsible for DCM transformation have remained elusive. In this study, we investigated the DCM catabolism of Dehalobacterium formicoaceticum strain EZ94, contained in an enriched culture, using a combination of biochemical approaches. Initially, enzymatic assays were conducted with cell-free protein extracts, after protein separation by blue native polyacrylamide gel electrophoresis. In the slices with the highest DCM transformation activity, a high absolute abundance of the methyltransferase MecC was revealed by mass spectrometry. Enzymatic activity assays with heterologously expressed MecB, MecC, and MecE from strain EZ94 showed complete DCM transformation only when all three enzymes were present. Our experimental results, coupled with the computational analysis of MecB, MecC, and MecE sequences, enabled us to assign specific roles in DCM transformation to each of the proteins. Our findings reveal that both MecE and MecC are zinc-dependent methyltransferases responsible for DCM demethylation and re-methylation of a product, respectively. MecB functions as a cobalamin-dependent shuttle protein transferring the methyl group between MecE and MecC. This study provides the first biochemical evidence of the enzymes involved in the anaerobic metabolism of DCM.IMPORTANCEDichloromethane (DCM) is a priority regulated pollutant frequently detected in groundwater. In this work, we identify the proteins responsible for the transformation of DCM fermentation in Dehalobacterium formicoaceticum strain EZ94 using a combination of biochemical approaches, heterologous expression of proteins, and computational analysis. These findings provide the basis to apply these proteins as biological markers to monitor bioremediation processes in the field.

Fe/GMP functional nanomaterial enhancing the denitrification efficiency by bi-signal regulation: Electron transfer and microbial community

A novel functional nanomaterial composed of guanosine monophosphate (GMP) and Fe enhanced denitrification efficiency by regulating electron transfer and microbial community. Fe/GMP enhanced nitrate (NO3−) degradation rates by 3.00-fold in serum vial batch experiments, with a rate constant of 17.39 mg/(L·h) in sequencing batch reactor. Fe/GMP-mediated interface promoted the secretion of redox-active substances in the extracellular polymeric substances to enhance the extracellular electron transfer. Specifically, Fe/GMP regulated electron transfer and metabolism activity by dynamic conversion of Fe3+/Fe2+ redox signal. Additionally, enzyme activity assays verified the optimized electron distribution function of Fe/GMP and thus enhanced intracellular electron transfer. High-throughput sequencing confirmed Fe/GMP selectively enriched microorganisms (especially Thauera 50.70 %). The tetraethylammonium stress experiment demonstrated Fe/GMP as an exogenous signaling molecule to restore microbial communication for microbial community regulation. The study proposes a multifaceted synergistic mechanism based on the repeater function of Fe/GMP in denitrification and offers insights for practical applications.

Elucidating the bioremediation potential of laccase and peroxidase enzymes from Bacillus ligniniphilus L1 in antibiotic degradation: A computationally guided study

This study showcased the antibiotic degradation abilities of laccase and catalase-peroxidase from Bacillus ligniniphilus L1, an extremophile, against 18 common antibiotics using computationally guided approach. Molecular docking and simulation identified six enzyme-antibiotic complexes for laccase and four for catalase-peroxidase, demonstrating significant binding affinity and stability. Enzyme activity assays corroborated computational results, indicating both enzymes could degrade all tested antibiotics with varying efficiencies. L1 laccase outperformed commercial laccase against five antibiotics, notably vancomycin, levofloxacin, tobramycin, linezolid, and rifamycin, with enhanced degradation potential upon ABTS addition. Catalase-peroxidase from L1 exhibited superior degradation efficiency over commercial peroxidase against vancomycin, linezolid, tobramycin, and clindamycin. Overall, this study underscores the computational approach’s utility in understanding enzyme-mediated antibiotic degradation, offering insights into environmental contaminant remediation.

Revealing synergistic mechanisms of biochar-assisted microbial electrolysis cells in enhancing the anaerobic digestion performance of waste activated sludge: Extracellular polymeric substances characterization, enzyme activity assay, and multi-omics analysis

Although biochar (BC)-assisted microbial electrolysis cells (MEC) has been shown to improve anaerobic digestion (AD) performance of waste activated sludge (WAS), the underlying mechanisms remain unclear. This study conducted an in-depth investigation into the mechanism based on analyses of extracellular polymeric substances (EPS) characteristics, enzyme activities and multi-omics. The results showed that compared with the control group, methane production improved by 16.73 %, 21.32 %, and 29.37 % in the BC, MEC, and BC-assisted MEC (BC-MEC) groups, respectively. The reconfiguration of the protein secondary structure increased the hydrophobicity of the EPS, thereby promoting microbial aggregation. In addition, partial least-squares path modeling (PLS-PM) and mantel test based on the enzyme activity and multi-omics analyses revealed that the promotional effect of MEC on the hydrolysis of WAS was superior to that of BC, while BC was more advantageous in promoting electron transfer and biofilm formation regulated by quorum sensing. The synergistic effects of BC and MEC were exemplified in the BC-MEC group. g_norank_Aminicenantales responsible for the hydrolysis of WAS was enriched (29.6 %), and the activities of hydrolytic enzymes including α-glucosidases and proteases were increased by 29.1 % and 43.6 %, respectively. Further, the expressions of genes related to acyl homoserine lactones (AHLs) and diffusible signal factor (DSF) in quorum sensing systems, as well as the genes related to hydrogenase involved in electron transfer (mbhJKL, hyfB-JR, hypA-F, and hoxFHUY), were up-regulated in the BC-MEC group. This facilitated electron transfer and microbial communication, consequently enhancing methane production. This research significantly advances the understanding of the mechanism by which BC-assisted MEC enhances AD performance and provides valuable insights into strategies for improving energy recovery from WAS.

The effects of all-trans retinoic acid on prednisolone-induced osteoporosis in zebrafish larvae

Glucocorticoids (GCs) are extensively used as anti-inflammatory and immunosuppressive medications in the long-term treatment of rheumatic disorders, respiratory diseases, renal diseases, and organ transplantation. Prolonged use of GCs can reduce bone mineral density, leading to osteoporosis (Glucocorticoid Induced Osteoporosis, GIOP) and fracture. All-trans retinoic acid (ATRA) is an active vitamin A metabolite that regulates embryonic development and adult organ function. ATRA has been found in studies to enhance osteogenesis. To examine the interventional effects of ATRA on GIOP and the mechanisms of ATRA activities, we first performed bioinformatic analysis to identify potential gene targets of ATRA. Zebrafish larvae were recruited as experimental animals, and the frequently used GC, prednisolone, was administered to larvae to construct a GIOP model. We evaluated the influence of exogenous ATRA on the activities of bone metabolic enzymes, the expression of genes linked to osteoblasts and osteoclasts, and the restoration of bone mineral density and bone mass in GIOP zebrafish larvae. Furthermore, we studied the influence of RBM14, a transcriptional coactivator and negative reciprocal factor of ATRA, on the regulation of osteoblastic gene expression during the anti-GIOP process of ATRA using the morpholino knockdown approach. The findings of bone metabolic enzyme activity (alkaline phosphatase, ALP and tartrate-resistant acid phosphatase, TRAP) and expression assays of osteoblastic marker genes (Runx2a, Runx2b, SP7, Csf1a, RANKL, and CTSK) indicated that ATRA had bidirectional effects on osteogenesis. However, in the GIOP model, ATRA reversed the GIOP-induced osteoporosis phenotype by inhibiting the GIOP-induced suppression of osteoblastic metabolic enzyme (ALP) activities and osteoblastic marker gene expression (Runx2a, Runx2b, and SP7), and this antagonism was concentration-dependent. We also observed that ATRA inhibited RBM14 expression in zebrafish larvae, while ATRA alone and RBM14 knockdown showed a consistent induction of osteoblast marker gene expression, implying that ATRA's inhibitory effect on RBM14 expression may underlie ATRA's osteogenic effects. Based on these data, we postulated that ATRA may ameliorate GIOP by decreasing RBM14 expression, thereby enhancing osteoblastic marker gene expression.

New mechanistic insights of nanoplastics synergistic cadmium induced overactivation of trypsin: Joint analysis from protein multi-level conformational changes and computational modeling

Nanoplastics (NPs) are emerging global contaminants that can exacerbate the animal toxicity and cytotoxicity of cadmium (Cd). However, the mechanisms by which NPs influence the toxic effects of Cd on key functional proteins within the body remain unknown. In this study, trypsin, a protein that is prone to coexist with NPs in the digestive tract, was selected as the target protein. The effects and mechanisms of NPs on Cd2+-induced structural damage at multiple levels and alterations in the biological function of trypsin were investigated using multi-spectroscopy techniques, enzyme activity assays, and computational modeling. Results indicated that the Cd2+-induced decrease and red shift of the trypsin backbone peak were exacerbated by the presence of NPs, leading to more serve backbone loosening. Furthermore, compared to Cd2+, NPs@Cd2+ caused a more pronounced reduction in the α-helix content of trypsin. These structural changes led to the opening of the trypsin pocket and the overactivation of the enzyme (NPs@Cd2+: 227.22%; Cd2+: 53.35%). Ultimately, the formation of a “protein corona” around NPs@Cd2+ and the metal contact of Cd2+ to the trypsin surface were identified as the mechanisms by which NPs enhanced the protein toxicity of Cd2+. This study elucidates, for the first time, the effects and underlying mechanisms of NPs on the toxicity of key functional proteins of Cd2+. These findings offer novel mechanistic insights and critical evidence essential for evaluating the risks associated with NPs.

Streptococcus suis 5’-nucleotidases contribute to adenosine-mediated immune evasion and virulence in a mouse model

ABSTRACT Streptococcus suis (S. suis) is an important swine bacterial pathogen and causes human infections, leading to a wide range of diseases. However, the role of 5’-nucleotidases in its virulence remains to be fully elucidated. Herein, we identified four cell wall-anchored 5’-nucleotidases (Snts) within S. suis, named SntA, SntB, SntC, and SntD, each displaying similar domains yet exhibiting low sequence homology. The malachite green reagent and HPLC assays demonstrated that these recombinant enzymes are capable of hydrolysing ATP, ADP, and AMP into adenosine (Ado), with the hierarchy of catalytic efficiency being SntC>SntB>SntA>SntD. Moreover, comprehensive enzymatic activity assays illustrated slight variances in substrate specificity, pH tolerance, and metal ion requirements, yet highlighted a conserved substrate-binding pocket, His–Asp catalytic dyad, metal, and phosphate-binding sites across Snts, with the exception of SntA. Through bactericidal assays and murine infection assays involving in site-mutagenesis strains, it was demonstrated that SntB and SntC collaboratively enhance bacterial survivability within whole blood and polymorphonuclear leukocytes (PMNs) via the Ado-A2aR pathway in vitro, and within murine blood and organs in vivo. This suggests a direct correlation between enzymatic activity and enhancement of bacterial survival and virulence. Collectively, S. suis 5’-nucleotidases additively contribute to the generation of adenosine, influencing susceptibility within blood and PMNs, and enhancing survival within blood and organs in vivo. This elucidation of their integral functions in the pathogenic process of S. suis not only enhances our comprehension of bacterial virulence mechanisms, but also illuminates new avenues for therapeutic intervention aimed at curbing S. suis infections.

Regulatory Network of the Late-Recruited Primary Decarboxylase C4NADP-ME in Sugarcane.

In agronomically important C4 grasses, efficient CO2 delivery to Rubisco is facilitated by NADP-malic enzyme (C4NADP-ME), which decarboxylates malate in bundle sheath cells. However, understanding the molecular regulation of the C4NADP-ME gene in sugarcane (Saccharum spp.) is hindered by its complex genetic background. Enzymatic activity assays demonstrated that decarboxylation in sugarcane Saccharum spontaneum predominantly relies on the NADP-ME pathway, similar to sorghum (Sorghum bicolor) and maize (Zea mays). Comparative genomics analysis revealed the recruitment of eight core C4 shuttle genes, including C4NADP-ME (SsC4NADP-ME2), in the C4 pathway of sugarcane. Contrasting to sorghum and maize, the expression of SsC4NADP-ME2 in sugarcane is regulated by different transcription factors (TFs). We propose a gene regulatory network for SsC4NADP-ME2, involving candidate TFs identified through gene co-expression analysis and yeast one-hybrid experiment. Among these, ABA INSENSITIVE5 (ABI5) was validated as the predominant regulator of SsC4NADP-ME2 expression, binding to a G-box within its promoter region. Interestingly, the core element ACGT within the regulatory G-box was conserved in sugarcane, sorghum, maize, and rice (Oryza sativa), suggesting an ancient regulatory code utilized in C4 photosynthesis. This study offers insights into SsC4NADP-ME2 regulation, crucial for optimizing sugarcane as a bioenergy crop.