Enzyme Function Research Articles

Synergistic Integration of a Ru(bda)‐Based Catalyst in a Covalent Organic Framework for Enhanced Photocatalytic Water Oxidation

AbstractTo address the urgent need for sustainable energy processes, there is a growing demand for multifunctional materials that mimic natural photosynthetic enzyme functions, specifically light‐harvesting, efficient photoinduced charge separation, and integration of molecularly defined catalysts, synergistically interacting within these structures. Herein, the successful synthesis of an innovative Covalent Organic Framework (COF‐TFPT‐IsoQ) constructed from optically active triazine (TFPT) and isoquinoline units (IsoQ) as building blocks is reported. Post‐synthetic incorporation of a Ru(bda)‐based water oxidation catalyst (WOC) is achieved through the IsoQ moieties acting as coordinating sites. Leveraging the synthetic flexibility of the designed COF architecture featuring binding sites on its pore walls, various Ru@COF‐TFPT‐IsoQ systems at different Ru:COF ratios are synthesized and tested in the photoinduced (λ > 400 nm) oxygen evolution reaction (OER) under sacrificial conditions. All synthesized Ru@COF‐TFPT‐IsoQ systems demonstrate efficiency in the photocatalytic OER, with the highest turnover number (TON) of 9.1 observed for the system where the Ru‐based WOC is incorporated every fourth COF‐TFPT‐IsoQ unit cell. This work provides valuable insights into the structural integration and catalytic behavior of Ru‐based complexes within COF architectures, highlighting the potential of Ru@COF‐TFPT‐IsoQ as a robust, efficient, and synthetically flexible multifunctional material for light‐induced water oxidation catalysis.

MetAP2 as a Therapeutic Target for Obesity and Type 2 Diabetes: Structural Insights, Mechanistic Roles, and Inhibitor Development

Type 2 Diabetes Mellitus (T2DM) and obesity are globally prevalent metabolic disorders characterized by insulin resistance, impaired glucose metabolism, and excessive adiposity. Methionine aminopeptidase 2 (MetAP2), an intracellular metalloprotease, has emerged as a promising therapeutic target due to its critical role in regulating lipid metabolism, energy balance, and protein synthesis. This review provides a comprehensive analysis of MetAP2, including its structural characteristics, catalytic mechanism, and functional roles in the pathophysiology of T2DM and obesity. The unique architecture of MetAP2’s active site and its interactions with substrates are examined to elucidate its enzymatic function. The review also explores the development of MetAP2 inhibitors, focusing on their mechanisms of action, preclinical and clinical findings, and therapeutic potential. Special emphasis is placed on docking studies to analyze the binding interactions of six key inhibitors (fumagillin, TNP-470, beloranib, ZGN-1061, indazole, and pyrazolo[4,3-b]indole) with MetAP2, revealing their structural determinants for efficacy and specificity. These findings underscore the potential of MetAP2 as a therapeutic target and provide valuable insights for the rational design of next-generation inhibitors to address obesity and T2DM.

Enhancing the reverse transcriptase function in Taq polymerase via AI-driven multiparametric rational design

IntroductionModification of natural enzymes to introduce new properties and enhance existing ones is a central challenge in bioengineering. This study is focused on the development of Taq polymerase mutants that show enhanced reverse transcriptase (RTase) activity while retaining other desirable properties such as fidelity, 5′- 3′ exonuclease activity, effective deoxyuracyl incorporation, and tolerance to locked nucleic acid (LNA)-containing substrates. Our objective was to use AI-driven rational design combined with multiparametric wet-lab analysis to identify and validate Taq polymerase mutants with an optimal combination of these properties.MethodsThe experimental procedure was conducted in several stages: 1) On the basis of a foundational paper, we selected 18 candidate mutations known to affect RTase activity across six sites. These candidates, along with the wild type, were assessed in the wet lab for multiple properties to establish an initial training dataset. 2) Using embeddings of Taq polymerase variants generated by a protein language model, we trained a Ridge regression model to predict multiple enzyme properties. This model guided the selection of 14 new candidates for experimental validation, expanding the dataset for further refinement. 3) To better manage risk by assessing confidence intervals on predictions, we transitioned to Gaussian process regression and trained this model on an expanded dataset comprising 33 data points. 4) With this enhanced model, we conducted an in silico screen of over 18 million potential mutations, narrowing the field to 16 top candidates for comprehensive wet-lab evaluation.Results and DiscussionThis iterative, data-driven strategy ultimately led to the identification of 18 enzyme variants that exhibited markedly improved RTase activity while maintaining a favorable balance of other key properties. These enhancements were generally accompanied by lower Kd, moderately reduced fidelity, and greater tolerance to noncanonical substrates, thereby illustrating a strong interdependence among these traits. Several enzymes validated via this procedure were effective in single-enzyme real-time reverse-transcription PCR setups, implying their utility for the development of new tools for real-time reverse-transcription PCR technologies, such as pathogen RNA detection and gene expression analysis. This study illustrates how AI can be effectively integrated with experimental bioengineering to enhance enzyme functionality systematically. Our approach offers a robust framework for designing enzyme mutants tailored to specific biotechnological applications. The results of our biological activity predictions for mutated Taq polymerases can be accessed at https://huggingface.co/datasets/nerusskikh/taqpol_insilico_dms

Effects of dietary synbiotic supplementation on digestive enzyme, total Vibrio count, and hepatopancreas of Pacific white shrimp, Penaeus vannamei challenged with Vibrio parahaemolyticus

ABSTRACT This study evaluated the effects of dietary synbiotics on enhancing digestive enzymes, reducing the overall bacterial count of Vibrio, and minimizing hepatopancreatic histological damage caused by V. parahaemolyticus in shrimp (Penaeus vanamei). The experiments involved administering different treatments, namely Bacillus NP5 (SBM), Pseudoalteromonas piscicida1Ub (SPM), and Bacillus NP5 and P. piscicida 1Ub (SBPM), incombination with the prebiotic mannan oligosaccharide (MOS). This study was conducted for 60 days, followed by a challenge test with Vibrio parahaemolyticus for 7 days. The results of the experiments showed that dietary synbiotic supplementation demonstrated better digestive enzyme activity and histology of the hepatopancreas compared to controls (p < .05). After the challenge test, it was found that the damage to the hepatopancreatic tissue of shrimps was less severe and the total vibrio count was lower in the synbiotic treatment, indicating a protective effect compared to the positive controls (p < .05). In conclusion, the use of dietary synbiotics had the potential to enhance digestive enzyme function and provide disease protection for Penaeus vannamei shrimp.

Overexpression of rice High-affinity Potassium Transporter gene OsHKT1;5 improves salinity and drought tolerance in Arabidopsis.

Rice is subjected to various environmental stresses, resulting in significant production losses. Abiotic stresses, particularly drought and salinity, are the leading causes of plant damage worldwide. The High-affinity Potassium Transporter (HKT) gene family plays an important role in enhancing crop stress tolerance by regulating physiological and enzymatic functions. This study investigates the effect of overexpressing the rice HKT1;5 gene in Arabidopsis thaliana on its tolerance to salinity and drought. The OsHKT1;5 gene was introduced into Arabidopsis under the control of 35S promoter of CaMV via floral dip transformation method. PCR confirmed the integration of the transgene into the Arabidopsis genome, while qPCR analysis showed its expression. Three transgenic lines of OsHKT1;5 were used for stress treatment and phenotypic studies. The overexpressed lines showed considerably higher germination rates, increased leaf counts, greater fresh and dry weights of the roots and shoots, higher chlorophyll contents, longer root lengths, and reduced Na+ levels together with increased K+ ions levels after salt and drought treatments, in comparison to wild-type plants. Furthermore, overexpressed lines exhibited higher antioxidant levels than wild-type plants under salinity and drought conditions. In addition, transgenic lines showed higher expression levels of the OsHKT1;5 gene in both roots and shoots compared to wild-type plants. In conclusion, this study revealed OsHKT1;5 as a promising candidate for enhancing tolerance to salinity and drought stresses in rice, marking a significant step toward developing a new rice variety with improved abiotic stress tolerance.

Two detoxification enzyme genes, CYP6DA2 and CarFE4, mediate the susceptibility to afidopyropen in Semiaphis heraclei

IntroductionSemiaphis heraclei is an important economic pest affecting Caprifoliaceae and Apiaceae plants, and chemical control is still the main effective control method in the field. Afidopyropen is a new type of pyridine cyclopropyl insecticide, which can effectively control piercing-sucking mouthparts pests and is suitable for pest resistance management. However, the detoxification mechanism of S. heraclei to afidopyropen is still poorly cleared.MethodsThe insecticidal activity of afidopyropen against S. heraclei and the enzyme activity assay and synergism bioassay were evaluated. The detoxification enzyme genes were obtained by transcriptome and validated by quantitative real-time PCR (RT-qPCR). Furthermore, RNA interference was used to study the functions of detoxification enzyme genes.ResultsThe activities of cytochrome P450 monooxygenases (P450s) and carboxylesterases (CarEs) were significantly increased under afidopyropen treatment. The toxicity of afidopyropen against S. heraclei was significantly increased after application the inhibitors of piperonyl butoxide and triphenyl phosphate. Sixteen P450 genes and three CarE genes were identified in the transcriptome of S. heraclei. The RT-qPCR results showed that eleven P450 genes and two CarE genes were significantly upregulated under afidopyropen treatment, and the expression of CYP6DA2 and CarFE4 was upregulated by more than 2.5 times. The expression pattern of CYP6DA2 and CarFE4 was further analyzed in different developmental stages of S. heraclei and knockdown of CYP6DA2 and CarFE4 significantly increased the susceptibility of S. heraclei to afidopyropen.ConclusionThe results of this study uncover the key functions of CYP6DA2 and CarFE4 in the detoxification mechanism of S. heraclei to afidopyropen, and provide a theoretical basis for the scientific use of afidopyropen in the field.

Effects of energetic compounds on soil microbial communities and functional genes at a typical ammunition demolition site.

High concentrations of energetic compounds such as 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) in military-contaminated sites pose a serious threat to human health and ecosystems. Better understanding about their effects on microbial diversity and functional genes in soil of ammunition demolition sites is required. In this study, the information of soil microbial community composition was obtained by macrogenome sequencing, and the impacts of energetic compounds on microbial community structure at the level of functional genes and enzymes based on Nr (Non-Redundant Protein Sequence Database), KEGG (Kyoto Encyclopedia of Genes and Genomes), CAZy (Carbohydrate-Active En-zymes Database) and other databases were discussed. The results showed that soil microbial diversity and functional gene abundance decreased significantly with the increase of the concentrations of energetic compounds. Conversely, the relative abundance of Proteobacteria increased significantly, reaching over 80% in the heavily TNT-contaminated area near explosive-waste water pool. Furthermore, functional gene analysis indicated that Proteobacteria had an advantage in degrading energetic compounds, and thus had the potential to improve the soil quality at ammunition demolition sites. This study provides a scientific basis for the future remediation and management of contaminated soils at ammunition demolition sites, as well as for the selection of efficient degraders of energetic compounds.

3D variability analysis reveals a hidden conformational change controlling ammonia transport in human asparagine synthetase

Advances in X-ray crystallography and cryogenic electron microscopy (cryo-EM) offer the promise of elucidating functionally relevant conformational changes that are not easily studied by other biophysical methods. Here we show that 3D variability analysis (3DVA) of the cryo-EM map for wild-type (WT) human asparagine synthetase (ASNS) identifies a functional role for the Arg-142 side chain and test this hypothesis experimentally by characterizing the R142I variant in which Arg-142 is replaced by isoleucine. Support for Arg-142 playing a role in the intramolecular translocation of ammonia between the active site of the enzyme is provided by the glutamine-dependent synthetase activity of the R142 variant relative to WT ASNS, and MD simulations provide a possible molecular mechanism for these findings. Combining 3DVA with MD simulations is a generally applicable approach to generate testable hypotheses of how conformational changes in buried side chains might regulate function in enzymes.

Identification and characterization of the cytochrome p450 complement in &lt;i&gt;Streptomyces cavourensis&lt;/i&gt; YBQ59

Cytochrome P450 enzymes (CYPs) are regarded as some of the most versatile biocatalysts. They are attractive candidates for natural product development because of their ability to selectively oxidize a broad range of substrates. Streptomyces spp. are not only producers of biologically active secondary metabolites but also a rich source of P450 enzymes. However, only a limited number of studies have explored the function and potential of P450 enzymes encoded in the Streptomyces genomes. In this study, the endophytic Streptomyces cavourensis YBQ59 isolated from Cinnamomum cassia J. Presl was sequenced using the Illumina sequencing platform to identify its P450 enzymes. The genome of YBQ59 was approximately 8,126,002 bp in size, with a G + C content of 72.1% and contained 7,020 genes. Genome annotation identified 21 CYP genes, distributed across 10 CYP families and 17 subfamilies. The possible role of these P450 enzymes in the synthesis of secondary metabolites was discussed. Since CYPs often require electron transport proteins to function, we analyzed the physical map of the genes encoding ferredoxins and ferredoxin reductases found in the genome of S. cavourensis YBQ59. Additionally, a phylogenetic tree was constructed to compare the P450 enzyme system from S. cavourensis YBQ59 with those of closely related and well-studied Streptomyces species, including Streptomyces sp. CFMR7, S. fulvissimus DSM 40593, S. griseus IFO 13350, and S. globisporus 1912. These results provide a basis for exploiting potential P450 enzymes from S. cavourensis YBQ59 for agricultural and medicinal applications.

Molecular insights into the aspartate protease Plasmepsin II activity inhibition by fluoroquinolones: A pathway to antimalarial drug development

Plasmepsin II (PlmII) belongs to the aspartate proteases and is involved in hemoglobin degradation in Plasmodium falciparum. Due to its critical role in the survival of the Plasmodium, PlmII is considered as a potent drug target for antimalarial therapy. We have done recombinant protein production of pro-plasmepsin II (Pro-plmII). Pro-PlmII was further activated to mature form (mPlmII) and its activity was confirmed by enzyme kinetics studies. The fluorescence spectroscopic and isothermal titration calorimetric studies show that fluoroquinolone-based antibiotic drugs norfloxacin, ciprofloxacin, and sparfloxacin bind with mPlmII. Molecular docking results show that only norfloxacin and ciprofloxacin are able to bind at the active site of mPlmII via hydrogen binding and hydrophobic interactions. Enzyme kinetics analysis reveals that norfloxacin and ciprofloxacin effectively inhibit mPlmII activity, while sparfloxacin does not exhibit any inhibitory effect on the enzyme's catalytic function. The two methyl groups on the 3rd and 5th carbon atoms of the piperazine ring make sparfloxacin unable to go inside mPlmII and bind at its active site. Overall, the results here suggested that fluoroquinolone-based antibiotic drugs norfloxacin, and ciprofloxacin, can be repurposed as antimalarial inhibitors targeting aspartic proteases. These findings contribute to pave the way for potential therapeutic interventions targeted at malaria.

Simultaneous nitrification and denitrification framework for decentralized systems: Long-term study utilizing rope-type biofilm media under field conditions

This research introduces a novel approach to achieve simultaneous nitrification-denitrification (SND) under dynamic load conditions using a cost-effective rope-type biofilm technology. The approach represents a significant advancement in wastewater treatment, particularly beneficial for remote and decentralized communities. The biofilm-based SND process was developed using a pilot-scale flow-through reactor by implementing upstream carbon management with constant-timer-based aeration control versus dynamic-sensor-based aeration control strategies. The findings indicate that adding an upstream anaerobic pretreatment process to handle excess carbon plays a substantial role in achieving a sustainable SND process under a dynamic load environment using simple aeration on-off control. The most optimal nitrification performance of 0.32 g NH3-N/m2/d (89 % removal) was achieved under a 1-hour ON/30-minute OFF aeration. The process sustained an average bulk liquid DO of 5.16 mg/L and 3.80 mg/L during the aeration ON and OFF periods, respectively, facilitating a 0.13 g N/m2/d (41 %) total inorganic nitrogen (TIN) removal, notably, implementing advanced aeration strategies driven by DO, NH3, and NO3 sensors enhanced TIN removal efficiency to 72 %. The nitrification performance remained comparable (89 % removal), resulting in 3 and 10 mg N/L effluent ammonia and TIN concentration, respectively. Additionally, utilizing two multivariate approaches accounting for 82 % and 64 % of the variance, this study discerned patterns in monitored variables and performance. Additionally, the analysis underscored the difference of bulk liquid DO levels in the biofilm versus suspended systems inhibiting the SND process. Distinct bacterial communities were established in biofilms under aerobic, anaerobic, and SND conditions, with the SND reactor showing a hierarchy of functional group and enzymes, enriched sequentially from heterotrophs to denitrifiers, nitrifiers, and anammox bacteria. These innovations underline the potential of tailored control strategies to enhance a passive biofilm-based SND process efficiency under dynamic conditions, providing scalable solutions for diverse target water quality demands in remote communities and decentralized systems.

Functional Profiling of Abundant Glycosyltransferases in the Rhizospheric Bacteriome of Abutilon fruticosum

In this study, we undertook an extensive exploration of the rhizospheric bacteriome of Abutilon fruticosum, focusing on the activities of Carbohydrate-Active Enzymes (CAZymes) of class glycosyltransferases (GTs), and their potential impact on plant growth and fortification against environmental adversities and soil-borne pathogens. Investigation revealed a predominance of families GT1, GT31, GT35, GT39, and GT51, mainly ascribed to bacterial genera Microvirga, Arthrobacter, Blastococcus, Bacillus, Nocardioides, and Kocuria. Our findings unveiled the pivotal role of phenylalanine, mostly exuded by plant roots, serving as the primary substrate for the catalytic activities of the identified CAZymes. With glucosylation emerging as the predominant enzymatic function, we propose UDP-D-glucose as the central core intermediate metabolite, orchestrating a multifaceted network of interrelated metabolic pathways. Subsequent profiling delineated a diverse array of metabolites synthesized by 10 CAZymes actively participating in 10 crosstalking pathways. Functional analyses of these metabolites underscore their significance in the biosynthesis of flavonoids, anthocyanins, and related derivatives, crucial for the reproductive success of plants by serving as visual cues for insect pollinators and conferring protection against deleterious effects of radiation. Certain other metabolites play vital roles in reinforcing the structural integrity of plant cell walls, thus enhancing resistance against enzymatic degradation while augmenting cytokinin activity. In conclusion, our study elucidates a novel paradigm of plant-bacteria mutualistic interaction, offering insights into the intricate mechanisms underlying plant-microbe symbiosis. We posit that the integration of high-throughput technologies with metabolic engineering strategies presents a promising approach for applying cost-effective and environmentally benign alternatives to conventional agricultural practices. (249 words)

Diagnosing Late-Onset Tay-Sachs Through Next Generation Sequencing and Functional Enzyme Testing

Diagnosing Late-Onset Tay-Sachs Through Next Generation Sequencing and Functional Enzyme Testing

Fine-Tuning the Function of Farnesene Synthases for Selective Synthesis of Farnesene Stereoisomers.

Farnesene synthase from Artemisia annua (AaFS) catalyzes the reaction from farnesyl pyrophosphate (FPP) to give the sesquiterpene β-farnesene, a key building block for the biosynthesis of vitamin E. However, an insufficient yield of β-farnesene precludes its industrialization. Understanding the mechanism would be essential for attaining β-farnesene in high yield. Guided by structure-based enzyme engineering, we designed several potent variants, among which L326I increased the β-farnesene yield from 450.65 to 3877.42 mg/L. Furthermore, we found that the function of β-farnesene synthase AaFS can be modulated at two positions; W299 is responsible for tuning the enzyme's function to give its isomeric product α-farnesene and Y402 is the key residue for diverting from the linear farnesene products to the monocyclic α-bisabolol product. These findings provide valuable insights into the catalytic mechanism and functional modulation of farnesene synthases and set the basis for rational engineering of farnesene synthases for selective biosynthesis of diverse sesquiterpene natural products.

Possible lessons of a model experiment: To what extent can UV activate the production of leaf phenolics in indoor plant cultivation?

Tobacco (Nicotiana tabacum L.) plants were grown outdoors (N°46.07, E°18.18) under either natural or UV-deprived sunlight for 25 days in the summer. High PAR resulted in high polyphenol content, which was selectively affected by solar UV-A and UV-B irradiation. Solar UV-A irradiation increased anthocyanins, but not flavonoids, in the epidermis, and this additional protection resulted in higher photochemical yields and lower NPQ. The simultaneous presence of UV-B overrode the effects of UV-A, increased epidermal flavonoids, and decreased anthocyanins. Leaves grown in full sunlight had the same photochemical yields of NPQ as those grown under a UV-excluding filter. A combination of these effects can falsely dismiss the effects of UV-B on outdoor photosynthesis. Phenolic acid content, corresponding to approximately 80% of phenolic compounds, did not depend on solar UV, and total flavonoids increased under full solar UV irradiation, but not under UV-A only. The polyphenol content in outdoor leaves also served as a reference point for an indoor experiment, which showed that even a short, 4-day exposure of low PAR grown plants to UV from an artificial source increased the amount of some, although not all, components close to or even above outdoor levels. In indoor leaves, a selective increase in quercetin glycosides (to 62–85% of outdoor levels) supports both enzymatic and non-enzymatic antioxidant functions, and the increase in crypto- and neochlorogenic acids (to 76% and 117% of outdoor levels, respectively) suggests a redistribution among biosynthesis pathways. These results demonstrate the potential and efficiency of cultivation systems without sunlight.

Structure and identification of the native PLP synthase complex from Methanosarcina acetivorans lysate.

Many protein-protein interactions behave differently in biochemically purified forms as compared to their in vivo states. As such, determining native protein structures may elucidate structural states previously unknown for even well-characterized proteins. Here, we apply the bottom-up structural proteomics method, cryoID, toward a model methanogenic archaeon. While they are keystone organisms in the global carbon cycle and active members of the human microbiome, there is a general lack of characterization of methanogen enzyme structure and function. Through the cryoID approach, we successfully reconstructed and identified the native Methanosarcina acetivorans pyridoxal 5'-phosphate (PLP) synthase (PdxS) complex directly from cryogenic electron microscopy (cryo-EM) images of fractionated cellular lysate. We found that the native PdxS complex exists as a homo-dodecamer of PdxS subunits, and the previously proposed supracomplex containing both the synthase (PdxS) and glutaminase (PdxT) was not observed in cellular lysate. Our structure shows that the native PdxS monomer fashions a single 8α/8β TIM-barrel domain, surrounded by seven additional helices to mediate solvent and interface contacts. A density is present at the active site in the cryo-EM map and is interpreted as ribose 5-phosphate. In addition to being the first reconstruction of the PdxS enzyme from a heterogeneous cellular sample, our results reveal a departure from previously published archaeal PdxS crystal structures, lacking the 37-amino-acid insertion present in these prior cases. This study demonstrates the potential of applying the cryoID workflow to capture native structural states at atomic resolution for archaeal systems, for which traditional biochemical sample preparation is nontrivial.IMPORTANCEArchaea are one of the three domains of life, classified as a phylogenetically distinct lineage. There is a paucity of known enzyme structures from organisms of this domain, and this is often exacerbated by characteristically difficult growth conditions and a lack of readily available molecular biology toolkits to study proteins in archaeal cells. As a result, there is a gap in knowledge concerning the mechanisms governing archaeal protein behavior and their impacts on both the environment and human health; case in point, the synthesis of the widely utilized cofactor pyridoxal 5'-phosphate (PLP; a vitamer of vitamin B6, which humans cannot produce). By leveraging the power of single-particle cryo-EM and map-to-primary sequence identification, we determine the native structure of PLP synthase from cellular lysate. Our workflow allows the (i) rapid examination of new or less characterized systems with minimal sample requirements and (ii) discovery of structural states inaccessible by recombinant expression.

Establishment and mechanism of thioacetamide-induced hepatopancreas injury model in red swamp crayfish Procambarus clarkii

The red swamp crayfish (Procambarus clarkii), widely farmed in China, frequently suffers from hepatopancreatic diseases due to the expansion and environmental degradation of aquaculture. Hepatopancreatic injury models are essential for studying disease mechanisms and treatments, yet few exist for crustaceans. This study aims to establish a hepatopancreatic injury model in P. clarkii using the hepatotoxic compound thioacetamide (TAA) and investigate the underlying damage mechanisms. P. clarkii were intramuscularly injected with TAA at concentrations of 0.2, 0.4, and 0.8 mg/g and observed at 6, 12, 24, and 48 h. The results showed that TAA at 0.2–0.8 mg/g significantly affected hepatopancreatic function and oxidative stress-related enzymes in P. clarkii. However, only 0.8 mg/g TAA produced a time-dependent damage effect suitable for a hepatopancreatic injury model. Histopathological analysis revealed that P. clarkii injected with 0.8 mg/g TAA exhibited key model features, including R cell adhesion and swelling, hepatic tubule rupture, and numerous small vacuoles. Therefore, 0.8 mg/g TAA intramuscular injection can effectively establish a hepatopancreatic injury model in P. clarkii. Transcriptomic and metabolomic analyses revealed that metabolic disruptions in energy, amino acid, lipid, and nucleotide pathways are central to TAA-induced hepatopancreatic injury in P. clarkii. CYP2L1X1, CYP2U1lX1, and CYP2D14l are likely key contributors to the hepatopancreatic injury induced by TAA in P. clarkii. The results of this study will provide support for further research on the mechanisms of hepatopancreatic diseases in freshwater crayfish and the development of preventive and therapeutic drugs.

The Protective Effects of Carvacrol Against Doxorubicin-Induced Cardiotoxicity In Vitro and In Vivo.

Doxorubicin (DOX) is a remarkable chemotherapeutic agent, however, its adverse effect on DOX-induced cardiotoxicity (DIC) is a rising concern. Recent research has identified carvacrol (CAR), an antioxidant and anti-inflammatory agent, as a promising natural compound for protecting against DIC. This study aims to investigate the potential cardioprotective effects properties of CAR in vitro and in vivo. The cardioprotective effect of CAR was assessed by pretreating H9c2 cells with non-toxic CAR for 24h, followed by co-treatment with DOX (10μM) for an additional 24h. The cell viability was determined using an MTT assay. For the in vivo study, male Sprague-Dawley rats (200-250g) were randomly divided into three groups: control, cardiotoxicity (DOX), and treatment (CAR + DOX) groups. CAR (50mg/kg, BW) was administered orally to the CAR + DOX groups for 14days. Then, a single dose of DOX (15mg/kg/i.p, BW) was administered on day 15 for DOX and CAR + DOX groups. The rats were allowed to recover for 3days before being sacrificed. Our results demonstrated that DOX (10µM) significantly reduced H9c2 cell viability by 50% (p < 0.0001), and CAR (0.067µM) protected H9c2 cells from DIC (p = 0.0045). In the rat model, CAR pretreatment effectively mitigated DOX-induced reductions in systolic pressure (p = 0.0007), pulse pressure (p = 0.0213), hypertrophy (p = 0.0049), and cardiac fibrosis (p = 0.0006). However, the pretreatment did not alter the heart function, oxidative stress, and antioxidant enzymes. In conclusion, our results indicate that CAR could potentially serve as an adjuvant to reduce cardiotoxicity by ameliorating myocardial fibrosis and hypertrophy.

Tracing the intraspecies expansion of glyoxalase genes and their expanding roles across the genus Oryza.

The genus Oryza is of utmost importance to human civilization as two of its species became agronomically productive and widely cultivated, and also because wild rice is a treasure trove of beneficial alleles that can be used for crop improvement. Most of the wild rice genotypes are known for their stress tolerance several times more than the domesticated rice varieties. In this study, we aimed to carry out an exhaustive genomic survey to identify glyoxalase I (GLYI) and glyoxalase II (GLYII) genes across the 11 rice genomes sequenced so far. Notably, we found the putatively functional metal-dependent GLYI and GLYII enzymes to be conserved throughout domestication and a few homologous pairs to have undergone beneficial mutations to drive positive selection, and thus, acquire newer functions. Interestingly, we also report four newly identified GLYII members in O. sativa subsp. japonica in addition to the three previously reported GLYII genes. The presence of different types of cis-elements in the promoter region of the glyoxalase genes gives insights into their role and regulation under various developmental processes besides stress adaptation. Publicly available data suggests the role of glyoxalase genes particularly in salinity stress in both wild and cultivated rice as is also confirmed through qRT-PCR. Interestingly, we found less accumulation of MG and concurrently higher enzymatic activity of GLYI and GLYII proteins in stressed seedlings of selected wild rice genotypes indicating that glyoxalases indeed contribute to the intrinsic stress tolerance of wild rice.

Effects of Vitamin E and/or selenium nanoparticles on organ histology, hemato-biochemical parameters, immunity, gene expression, and growth performance in Nile tilapia challenged with Enterococcus faecalis

A two-month feeding experiment was conducted to assess the impact of dietary selenium nanoparticles (SeNPs), sodium selenite (Se), and vitamin E (Vit E) on the growth performance, hematology profile, innate immune response, histomorphology of the liver, intestine, and gills, as well as gene expression in Nile tilapia (Oreochromis niloticus) challenged with the pathogenic bacteria Enterococcus faecalis. A total of 360 fish were allocated into six experimental groups and fed diets containing 1 mg/kg of SeNPs (SeNPs), 1 mg /kg of sodium selenite (Se), 100 mg/kg of Vit E (VE), SeNPs+ Vit E, Se+ Vit E and a control group (CON) without any additives. The results showed that SeNPs and SeNPs+ Vit E diets significantly improved the growth performance (final body weight, feed intake, fish biomass, specific growth rate, and feed conversion ratio) and blood parameters compared to the other groups (P<0.05). Liver enzymes (AST, ALT, and ALP) were significantly decreased, while immune responses were enhanced in the serum of fish in the SeNPs and SeNPs+ Vit E groups before and after the E. faecalis challenge. The expression of pro-inflammatory genes (TNF-α and IL-8) was significantly lower in the SeNPs and SeNPs+ Vit E groups, both before and after the E. faecalis challenge, compared to the other groups (P<0.05). Nile tilapia fed the control diet and challenged with E. faecalis exhibited a high mortality rate (85 %), whereas mortality was significantly lower in fish fed supplemental SeNPs (45 %) and SeNPs+ Vit E (40 %). Histological examination also revealed improvements in liver, intestine, and gill functions in Nile tilapia fed SeNPs+ Vit E. These results suggest that dietary SeNPs, either alone or in combination with Vit E, can significantly enhance fish performance, innate immune response, liver function enzymes, and reduce the expression of pro-inflammatory genes in Nile tilapia. They can also mitigate the pathological effects caused by the E. faecalis challenge.