Enzyme Activity Assays Research Articles

Genome-wide investigation of family‑1 UDP glycosyltransferases reveals the Fusarium resistance role of VfUGT90A2 in tung trees

UDP-glycosyltransferases (UGTs) play vital roles in pathogen resistance through secondary metabolites, such as flavonoid. Vernicia fordii (tung trees) is well known for its production of industrial oil, which has been decimated by the soil-borne fungus Fusarium oxysporum f. sp. Fordiis (Fof-1). The current understanding of the metabolic and molecular mechanism of V. fordii against Fof-1 is limited. Through genome-wide analysis identified 153 VfUGTs, which were divided into 16 clusters derived from tandem and segmental repeat events. Variations in gene structure and cis-elements have contributed to gene differentiation within the various subfamilies of VfUGTs. VfUGT90A2 was found to be highly induced and expressed, serving as a key gene during the response process to Fof-1 infection in tree roots. The study showed that transgenic hairy roots of the tung trees, overexpressing VfUGT90A2, displayed significantly enhanced resistance to Fof-1. Additionally, the root extract from these transgenic roots was found to have a notable inhibitory effect on the growth of Fof-1 mycelium. Metabolomic and transcriptomic analyses further revealed an increase in flavonoids, such as quercitrin and myricitrin, in the transgenic hairy roots overexpressing VfUGT90A2. Meanwhile, VfUGT90A2 was speculated bound well to quercetin by protein docking predictions. The enzyme activity assay in vitro further verified that VfUGT90A2 catalyzed the glycosylation of quercetin. In all, our data shows that VfUGT90A2 is involved in the flavonoid biosynthetic pathways and has an impact on enhancing the resistance of plant to Fusarium, therefore, offers an opportunity to explore an effective strategy for Fusarium resistant plant breeding.

Genome-wide identification and expression analysis of the GT8 gene family in tomato(Solanum lycopersicum) and the functional of SlGolS1 under cold stress

Glycosyltransferases (GTs) are a diverse superfamily of enzymes involved in glycosylation reactions, with the GT8 (glycosyltransferase 8) playing a crucial role in plant growth, development, and abiotic stress responses. Tomato, widely cultivated and is a thermophilic plant. So it is significant to study how GT8 regulates cold resistance in tomato for plant growth. In this study, we screened the whole genome of tomato by using bioinformatics methods and identified 40 members of the GT8 gene family. Analysis of cold stress transcriptome data and qRT-PCR experiments revealed the potential significance of SlGolS1 in responding to cold stress. SlGolS1 was highly expressed in the stems and flowers of tomato, with its mature protein localized in the chloroplast. Used the VIGS method to transiently silence the SlGolS1 gene, the SlGolS1-silenced plants (pTRV2-SlGolS1) rendered tomato more sensitive to cold stress compared with the control (pTRV2) tomato plant phenotype after cold treatment; enzyme activity assays showed that oxidative damage was more severe in the pTRV2-SlGolS1. In summary, SlGolS1 positively regulates cold resistance in tomato.

Up-regulated succinylation modifications induce a senescence phenotype in microglia by altering mitochondrial energy metabolism.

The aging of the central nervous system(CNS) is a primary contributor to neurodegenerative diseases in older individuals and significantly impacts their quality of life. Neuroinflammation, characterized by activation of microglia(MG) and release of cytokines, is closely associated with the onset of these neurodegenerative diseases. The activated status of MG is modulated by specifically programmed metabolic changes under various conditions. Succinylation, a novel post-translational modification(PTM) mainly involved in regulating mitochondrial energy metabolism pathways, remains unknown in its role in MG activation and aging. In the present study, we found that succinylation levels were significantly increased both during aging and upon lipopolysaccharide-induced(LPS-induced) MG activation undergoing metabolic reprogramming. Up-regulated succinylation induced by sirtuin 5 knockdown(Sirt5 KD) in microglial cell line BV2 resulted in significant up-regulation of aging-related genes, accompanied by impaired mitochondrial adaptability and a shift towards glycolysis as a major metabolic pathway. Furthermore, after LPS treatment, Sirt5 KD BV2 cells exhibited increased generation of reactive oxygen species(ROS), accumulation of lipid droplets, and elevated levels of lipid peroxidation. By employing immunoprecipitation, introducing point mutation to critical succinylation sites, and conducting enzyme activity assays for succinate dehydrogenase(SDH) and trifunctional enzyme subunit alpha(ECHA), we demonstrated that succinylation plays a regulatory role in modulating the activities of these mitochondrial enzymes. Finally, down-regulation the succinylation levels achieved through administration of succinyl phosphonate(SP) led to amelioration of MG senescence in vitro and neuroinflammation in vivo. To our knowledge, our data provide preliminary evidence indicating that up-regulated succinylation modifications elicit a senescence phenotype in MG through alterations in energy metabolism. Moreover, these findings suggest that manipulation of succinylation levels may offer valuable insights into the treatment of aging-related neuroinflammation.

A comprehensive analysis of the defense responses of Odontotermes formosanus (Shiraki) provides insights into the changes during Serratia marcescens infection

BackgroundOdontotermes formosanus (Shiraki) is a highly damaging agroforestry pest. Serratia marcescens is a broad-spectrum insecticidal pathogen and is highly lethal to O. formosanus. However, little is known about the mechanism between them. To improve the biological control of pests, a more in-depth analysis of the interactions between the pests and the pathogens is essential.ResultsWe used RNA-seq, enzyme activity assays and real-time fluorescent quantitative PCR (qPCR) to explore the defense responses of O. formosanus against SM1. RNA-seq results showed that 1,160, 2,531 and 4,536 genes were differentially expressed at 3, 6 and 12 h after SM1 infection, and Kyoto Encyclopedia of Genes and Genomes (KEGG) results indicated that immune response and energy metabolism were involved in the defense of O. formosanus against SM1. Reactive oxygen species (ROS) levels and ROS synthesis genes were significantly elevated, and the antioxidant system were induced in O. formosanus after SM1 infection. In addition, the cellular immune genes were affected, and the Toll, immune deficiency (Imd), Janus kinase/signal transducer and activator of transcription (JAK/STAT), c-Jun N-terminal Kinase (JNK) and melanization pathways were activated. In vitro, Oftermicin, an antimicrobial peptide, had a significantly inhibitory effect on SM1. Furthermore, the expression levels and enzyme activities of phosphofructokinase (PFK), lactate dehydrogenase (LDH), succinate dehydrogenase (SDH) and isocitrate dehydrogenase (IDH) in glycolysis and tricarboxylic acid (TCA) cycles were increased.ConclusionsOur results clearly demonstrated that O. formosanus defended against SM1 by activating the antioxidant system, innate immunity and energy metabolism. This study would provide useful information for the development of biological controls of O. formosanus.

A Detailed Proteomics and Metabolomics Landscape Sheds Light on the Mechanistic Insights Into the Resistance Response of Transgenic Pigeon Pea Against Wilt Stress.

Pigeon pea, vital for farmers in semi-arid regions, suffers yield losses from Fusarium wilt caused by Fusarium udum. This study demonstrates that introducing the rice oxalate oxidase 4 (Osoxo4) gene significantly boosts wilt resistance. Enhanced resistance in transgenic lines was confirmed through gene expression analysis, enzyme activity assays, biochemical assessments, histochemical staining and in vitro and in vivo bioassays, including spore germination tests. We performed proteomics and metabolomics analyses to investigate mechanisms of enhanced resistance. LC-MS/MS-based label-free proteomics of wilt-infected transgenic and wild-type pigeon pea leaves identified 2386 proteins, with 1048 showing significant abundance changes-738 upregulated and 310 downregulated-in transgenic plants. Notably, proteins such as HMG1/2-like protein, Putative nucleosome assembly protein C364.06, DEAD-box ATP-dependent RNA helicase 3, Lipoxygenase 1, Annexin D1 and Annexin-like protein RJ4 were significantly upregulated, indicating their potential role in developing wilt-resistant cultivars. Metabolomic analysis showed elevated levels of amino acids, sugars, oxalic acid, sugar alcohols and myo-inositol in transgenic pigeon pea, with upregulated pathways in Sugar and Starch Metabolism and Inositol Phosphate Metabolism, indicating enhanced resilience to wilt stress. This study highlights unique regulatory proteins and metabolites, offering insights into stress adaptation and guiding genetic interventions for breeding disease-resistant pigeon pea varieties.

Metabolomic and enzymatic markers reveal critical air exposure threshold for Crassostrea hongkongensis quality

Post-harvest air exposure is unavoidable during oyster transportation and storage, yet the physiological tolerance limits and underlying metabolic responses of commercially important oyster species remain poorly understood. While previous studies have focused on immediate post-harvest quality changes, there is limited knowledge about the time-dependent metabolic adaptations that determine product quality during extended air exposure. This study investigated the physiological and metabolic responses of Crassostrea hongkongensis during air exposure at 4 °C, focusing on identifying the optimal period for quality preservation. Using a combination of survival analysis, enzyme activity assays, and metabolomic profiling, we examined oysters exposed to air for up to 18 days, with particular emphasis on the critical first three days. Survival analysis showed 100 % survival rate at 4 °C through day 7, with mortality beginning thereafter, compared to significant mortality observed at 25 °C (complete mortality by day 7) and 37 °C (complete mortality by day 2). Analysis of antioxidant enzyme activities revealed complex, time-dependent changes, with robust responses observed within the first three days, indicating effective stress management. Metabolomic analysis identified 38 differentially abundant metabolites throughout the exposure period. Notably, the metabolic profile at day 3 showed a tendency to revert towards the control state, suggesting a temporary adaptive response. Key findings included stability in total antioxidant capacity (T-AOC) levels during the initial three days and subtle changes in flavor-related compounds, such as slight decreases in glutamate and aspartate levels. Correlation analyses revealed intricate interactions between enzyme activities and metabolites, highlighting complex stress response mechanisms. The relationship between T-AOC and key osmolytes underscored their critical role in maintaining cellular redox balance during the initial exposure period. Our findings suggest that the optimal window for maintaining C. hongkongensis quality during air exposure at 4 °C is within the first three days. During this period, oysters demonstrate effective adaptive responses, maintaining key quality attributes and nutritional value. Beyond this timeframe, the risk of quality degradation increases significantly. These results have important implications for the oyster industry, providing evidence-based guidelines for post-harvest handling, transportation, and storage practices. We recommend limiting air exposure during cold storage to no more than 3 days to ensure optimal product quality and consumer satisfaction.

Hepatic enzyme induction and its potential effect on thyroid hormone metabolism in the metamorphosing tadpole of Xenopus laevis (African clawed frog).

Hepatic enzyme induction, an inherent defense system against xenobiotics, is known to simultaneously affect endocrine system functions in mammals under specific conditions, particularly thyroid hormone (TH) regulation. While this phenomenon has been studied extensively, the pathway leading to this indirect thyroid effect in mammals has unclear applicability to amphibians, despite the importance of amphibian species in assessing thyroid-disruptive chemicals. Here, we investigated the effects of three well-known mammalian enzyme inducers-β-naphthoflavone (BNF), pregnenolone carbonitrile (PCN), and sodium phenobarbital (NaPB)-on the gene expression of phase-I and phase-II metabolizing enzymes in Xenopus laevis tadpoles. Waterborne exposure to BNF and PCN significantly induced the expression of both phase-I (cytochrome P450, CYP) and phase-II enzymes (UDP-glucuronosyltransferase, UGT and sulfotransferase, SULT), but in different patterns, while NaPB exposure induced CYP2B expression without affecting phase-II enzymes in tadpoles, in contrast to mammals. Furthermore, an ex vivo hepatic enzyme activity assay confirmed that BNF treatment significantly increased phase-II metabolic activity (glucuronidation and sulfation) toward TH. These results suggest the potential for certain mammalian enzyme inducers to influence TH clearance in X. laevis tadpoles. Our findings provide insights into the profiles of xenosensing activity and enzyme induction in amphibians, which can facilitate a better understanding of the mechanisms of indirect effects on the thyroid system via hepatic enzyme induction in nonmammalian species.

Evaluating the estrogen degradation potential of laccase and peroxidase from Bacillus ligniniphilus L1 through integrated computational and experimental approaches

This study investigated the degradation potential of laccase and catalase-peroxidase from the extremophilic marine bacterium Bacillus ligniniphilus L1 against endogenous and synthetic estrogen compounds using an integrated computational and experimental approach. Molecular docking identified five estrogen compounds exhibiting reliable bindings with enzymes, which were then subjected to enzyme activity assays. The degradation potential of the two enzymes against five selected estrogen compounds were investigated and compared with their commercial counterparts. Laccase from L1 showed higher degradation potential against estrone (47.02 % without and 62.21 % with ABTS) compared to commercial laccase (39 % without and 54.20 % with ABTS). For estradiol valerate, commercial laccase showed a slightly higher degradation (52.47 %) than L1 laccase (49.94 %), but with ABTS, L1 laccase performed better (74.15 % vs. 68.03 %). Notably, L1 catalase-peroxidase demonstrated significantly higher degradation for all tested compounds compared to its commercial counterpart with efficiencies of 96.16 %, 89.09 %, 74.94 %, 64.91 %, and 62.80 % against estropipate, quinestrol, estradiol valerate, estriol and estrone, respectively, revealing its potential for commercial applications. Molecular dynamics simulations revealed the interaction and stability of enzyme-estrogen complexes, with MMGBSA binding energy calculations supporting experimental results. These findings highlight the usefulness of the computational approach in elucidating the molecular mechanisms underlying enzyme-mediated bioremediation of environmental contaminants.

Efficient removal of nitrate and ammonium by strain Pseudomonas poae EH-E3 under acidic pH and low-temperature conditions

The combined effect of low temperature and an acidic environment may restrict the growth and metabolic activity of microorganisms involved in nitrogen removal. To overcome poor biological nitrogen removal efficiency during low-temperature treatment of acidic wastewater, a novel pure strain of Pseudomonas poae EH-E3 was successfully isolated and screened based on its strong heterotrophic nitrification and aerobic denitrification capacity. Strain EH-E3 could completely eliminate inorganic nitrogen sources of nitrate within 30h and ammonium within 20h at 15 °C and pH 5.0, with corresponding removal efficiencies for total nitrogen of 95.13% and 91.35%, respectively. Nitrogen balance testing indicated that strain EH-E3 removed 43.25% of ammonium, 45.72% of nitrate, and 51.20% of nitrite in the form of gaseous products. Enzyme activity assays revealed that the specific activities of ammonia monooxygenase, nitrate reductase, and nitrite reductase were 0.533, 0.956, and 0.011 U/mg proteins, respectively. These findings suggest that strain EH-E3 has strong potential for use in low-temperature treatment of acidic wastewater.

Perturbation of Enzyme Structure by nano-Metal Organic Frameworks: A Question Mark on their Safety-by-Design?

Our study investigates the interactions between nanoscale Metal-Organic Frameworks (nMOFs), specifically ZIF-8 and CuIm, and key enzymes: Acetylcholine Esterase (AChE), α-amylase. Using circular dichroism (CD) spectroscopy, we observed significant alterations in the secondary structures of these enzymes upon interaction with nMOFs. AChE showed a reduction in α-helix content from 20.1% to a significantly lower value when exposed to 160µg/mL of nMOFs, with a corresponding increase in β-sheet and other structural components. Enzymatic activity assays revealed that CuIm nMOFs decreased AChE activity by 67.08% at the highest concentration tested (160µg/mL). ZIF-8 also affected AChE activity significantly at this concentration. Similarly, α-amylase exhibited structural changes, with increasing concentrations of nMOFs leading to a near-total loss of secondary structure at 80 and 160µg/mL. These structural changes were accompanied by a marked decrease in enzymatic activity, particularly with CuIm nMOFs showing the most substantial inhibitory effects. Our findings highlight the profound impact of nMOFs on enzyme structure and function, emphasising the need for comprehensive assessments of nMOFs' potential toxicity and understanding the aspects of their safety-by-design.

Molecular Profiling of Rice Straw Degradability Discrepancy in Stropharia rugosoannulata Core Germplasm.

The rice-S. rugosoannulata pattern is a rapidly growing agricultural practice for straw disposal and mushroom production in China. However, different S. rugosoannulata strains show a large variation in rice straw degradability. Here, we constructed a core collection of S. rugosoannulata containing 14 strains with rich genetic diversity. The molecular profiling of the lignocellulose degradability discrepancy of S. rugosoannulata strains was then explored using enzyme activity assays and transcriptome analysis. The results indicated that mycelial growth rate, lignocellulolytic enzyme activities, and rice straw degradability differed widely among the S. rugosoannulata core strains. The genes encoding lignin modifying and degrading auxiliary enzymes, oxidases, glycoside hydrolases, and detoxification proteins were differentially expressed between two representative S. rugosoannulata strains, resulting in differences in their lignocellulolytic enzyme activities and further causing differences in lignocellulose degradability. This study is useful to improve the production efficiency of S. rugosoannulata and promote the recycling of rice straw.

Astragaloside IV reduces mutant Ataxin-3 levels and supports mitochondrial function in Spinocerebellar Ataxia Type 3

This study investigated the therapeutic effects of astragaloside IV (AST) on spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), a neurodegenerative disorder. Human neuroblastoma SK-N-SH cells expressing mutant ataxin-3 protein with 78 CAG repeats (MJD78) were employed as an in vitro model. Protein expression analysis demonstrated that AST treatment reduced mutant ataxin-3 protein expression and aggregation by enhancing the autophagic process in MJD78 cells. Elevated oxidative stress levels in MJD78 cells were significantly reduced following AST treatment, which also enhanced antioxidant capacity, as evidenced by flow cytometry and antioxidant enzyme activity assays. Furthermore, AST treatment ameliorated mitochondrial dysfunction in MJD78 cells, including improvements in mitochondrial membrane potential, respiration, and mitochondrial dynamics. In conclusion, AST administration increased antioxidant capacity, reduced both cellular and mitochondrial oxidative stress, and improved mitochondrial quality control processes through fusion, fission, and autophagy. These mechanisms collectively reduced intracellular mutant ataxin-3 protein aggregation, thereby achieving therapeutic efficacy in the SCA3 model.

S-nitrosylation of aconitase-2 facilitates coronary microembolization-induced cardiac injury by suppressing iron-sulfur cluster assembly

Abstract Background Coronary microembolization (CME) is a common reason for periprocedural myocardial infarction after coronary interventions. S-nitrosylation (SNO), a prototypic redox-based posttranslational modification, is involved in the pathogenesis of several cardiovascular diseases. Purpose To explore the role of SNO of aconitase-2 (ACO2) in CME-induced cardiac injury, as well as the mechanism by which SNO-ACO2 modulates myocardial injury in response to CME. Methods CME models were established in C57BL/6J mice by injecting 300,000 polyethylene microspheres (diameter 9 µm) into the left ventricle chamber with the occlusion of the ascending aorta. Cardiac function was evaluated by echocardiography. Histological and serum analysis was performed to evaluate myocardial injury. Myocardial samples were scanned for S-nitrosylated proteins using biotin-switch procedures and liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. SNO sites of ACO2 were further identified through LC-MS/MS. De-nitrosylation of ACO2 was achieved by the mutation of SNO site or overexpression of the de-nitrosylation enzyme thioredoxin 1 (TRX1). Interacting effectors of SNO- ACO2 were screened through LC-MS/MS and confirmed by coimmunoprecipitation. Neonatal rat ventricular myocytes (NRVMs) were used for in vitro study. The cellular ACO2 activity was analysed using an ACO2 enzyme activity assay kit. Mitochondrial morphology and function were assessed by fluorescent staining and mitochondrial stress testing. Results Cardiac injury was exacerbated by CME as demonstrated by impaired cardiac contractile function, morphological changes, and increased fibrosis, microinfarct size and serum troponin I level. We identified 789 S-nitrosylated proteins in CME mouse hearts, and ACO2 is one of the highly S-nitrosylated proteins. The increased level of SNO-ACO2 was verified by immunoblot assay in CME mouse hearts and hypoxia-stimulated NRVMs. SNO site of ACO2 at cysteine 126 was identified by LC-MS/MS and confirmed in NRVMs. De-nitrosylation of ACO2 by the mutation of cysteine 126 or overexpression of TRX1 both greatly improved the activity of ACO2 by 39.7% and 31.4% respectively, furthermore restored the mitochondrial function, and attenuated CME-induced cardiac injury. Mechanistically, SNO-ACO2 at cysteine 126 suppressed the interaction between ACO2 and NFU1, an iron-sulfur (Fe-S) cluster scaffold protein. The dissociation from NFU1 deprived ACO2 of the Fe-S cluster and the enzyme activity, thereby promoting the disruption of the mitochondrial tricarboxylic acid cycle and cardiac injury. Conclusions Our data defined a previously unrecognized role of SNO-ACO2 in CME-induced cardiac injury. We demonstrated that SNO-ACO2 at cysteine 126 disrupted ACO2/NFU1 interaction to exacerbate myocardial injury after CME. Our results suggested that de-nitrosylation of ACO2 may provide a new therapeutic target for the treatment of CME.

The thylakoid phosphatase TEF8 is involved in state transition and high light stress resistance in Chlamydomonas.

The sophisticated regulation of state transition is required to maintain optimal photosynthetic performance under fluctuating light condition, through balancing the absorbed light energy between photosystem II andphotosystem I. This exquisite process incorporates phosphorylation and dephosphorylation of light-harvesting complexes and PSII core subunits, accomplished by thylakoid membrane-localized kinases and phosphatases that have not been fully identified. In this study, one Chlamydomonas high light response gene, THYLAKOID ENRICHED FRACTION 8 (TEF8), was characterized. The Chlamydomonas tef8 mutant showed high light sensitivity and defective state transition. The enzymatic activity assays showed that TEF8 is a bona fide phosphatase localized in thylakoid membranes. Biochemical assays, including BN-PAGE, pull-down, and phosphopeptide mass spectrometry, proved that TEF8 associates with photosystem II and is involved in the dephosphorylation of D2 and CP29 subunits during state 2 to state 1 transition. Taken together, our results identified TEF8 as a thylakoid phosphatase with multiple dephosphorylation targets on photosystem II, and provide new insight into the regulatory mechanism of state transition and high light resistance in Chlamydomonas.

Reductive dehalogenase of Dehalococcoides mccartyi strain CBDB1 reduces cobalt- containing metal complexes enabling anodic respiration.

Microorganisms capable of direct or mediated extracellular electron transfer (EET) have garnered significant attention for their various biotechnological applications, such as bioremediation, metal recovery, wastewater treatment, energy generation in microbial fuel cells, and microbial or enzymatic electrosynthesis. One microorganism of particular interest is the organohalide-respiring bacterium Dehalococcoides mccartyi strain CBDB1, known for its ability to reductively dehalogenate toxic and persistent halogenated organic compounds through organohalide respiration (OHR), using halogenated organics as terminal electron acceptors. A membrane-bound OHR protein complex couples electron transfer to proton translocation across the membrane, generating a proton motive force, which enables metabolism and proliferation. In this study we show that the halogenated compounds can be replaced with redox mediators that can putatively shuttle electrons between the OHR complex and the anode, coupling D. mccartyi cells to an electrode via mediated EET. We identified cobalt-containing metal complexes, referred to as cobalt chelates, as promising mediators using a photometric high throughput methyl viologen-based enzyme activity assay. Through various biochemical approaches, we show that cobalt chelates are specifically reduced by CBDB1 cells, putatively by the reductive dehalogenase subunit (RdhA) of the OHR complex. Using cyclic voltammetry, we also demonstrate that cobalt chelates exchange electrons with a gold electrode, making them promising candidates for bioelectrochemical cultivation. Furthermore, using the AlphaFold 2-calculated RdhA structure and molecular docking, we found that one of the identified cobalt chelates exhibits favorable binding to RdhA, with a binding energy of approximately -28 kJ mol-1. Taken together, our results indicate that bioelectrochemical cultivation of D. mccartyi with cobalt chelates as anode mediators, instead of toxic halogenated compounds, is feasible, which opens new perspectives for bioremediation and other biotechnological applications of strain CBDB1.

Isolation and identification of cellulose decomposition bacteria from the soil of Yunnan province, China

In this experiment sodium carboxymethyl cellulose (CMC-Na) was used as the only carbon source to isolate cellulose decomposing bacteria from the soil of Qiongzhusi Forest Park in Kunming, Yunnan Province. The ratio of hydrolytic circle diameter and colony diameter formed by the strain on cellulose Congo red medium was determined for primary screening, and then the filter paper disintegration test and CMC enzyme activity assay were used for rescreen. A cellulose decomposing bacterium (HS-2) with high enzyme activity was obtained, and its enzyme activity was up to 16.42 U/ml. (U means the unit of activity of enzymes, activity, unit) Gram stain and Spore stain reaction showed that HS-2 strain was Gram-positive Bacillus. The Bacillus strain was identified as Bacillus pumilus on the basis of physiological and biochemical tests, and 16S rDNA sequence analysis. Bangladesh J. Bot. 52(3): 635-641, 2024 (September) Special

Modulation of Carnitine Palmitoyl Transferase 1b Expression and Activity in Muscle Pathophysiology in Osteoarthritis and Osteoporosis

In the pathophysiology of osteoarthritis and osteoporosis, articular cartilage and bone represent the target tissues, respectively, but muscle is also involved. Since many changes in energy metabolism occur in muscle with aging, the aim of the present work was to investigate the involvement of carnitine palmitoyl transferase 1b (Cpt1b) in the muscle pathophysiology of the two diseases. Healthy subjects (CTR, n = 5), osteoarthritic (OA, n = 10), and osteoporotic (OP, n = 10) patients were enrolled. Gene expression analysis conducted on muscle and myoblasts showed up-regulation of CPT1B in OA patients; this result was confirmed by immunohistochemical and immunofluorescence analyses and enzyme activity assay, which showed increased Cpt1b activity in OA muscle. In addition, CPT1B expression resulted down-regulated in cultured OP myoblasts. Given the potential involvement of Cpt1b in the modulation of oxidative stress, we investigated ROS levels, which were found to be lower in OA myoblasts, and gene expression of nicotinamide adenine dinucleotide phosphate hydrogen oxidase 4 (Nox4), which resulted up-regulated in OA cells. Finally, the immunofluorescence of BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (Bnip3) showed a decreased expression in OP myoblasts, with respect to CTR and OA. Contextually, through an ultrastructural analysis conducted by Transmission Electron Microscopy (TEM), the presence of aberrant mitochondria was observed in OP muscle. This study highlights the potential role of Cpt1b in the regulation of muscle homeostasis in both osteoarthritis and osteoporosis, allowing for the expansion of the current knowledge of what are the molecular biological pathways involved in the regulation of muscle physiology in both diseases.

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.