Molecular Techniques Research Articles

Geographical distribution and molecular identification of cyst nematodes in Bajaur, Pakistan

Summary A survey conducted along the Panjkora river in Bajaur, Khyber Pakhtunkhwa (KPK), Pakistan, identified seven isolates of nematode populations belonging to the Heteroderidae family among the 25 soil and root samples examined. Within these isolates, three were classified as Heterodera avenae, H. filipjevi and H. zeae, while the remaining four were identified as Globodera pallida. The identification of these cyst nematodes involved a combination of morphological and morphometric analyses, complemented by molecular techniques employing newly designed primers to amplify diagnostic regions from any cyst nematode. This study marks the first molecular characterisation of H. avenae, H. zeae and G. pallida in Pakistan and the initial report of H. filipjevi in the country. The mapping of crop-growing regions with sampling points highlights areas at high risk for further pathogen spread.

Network pharmacology-based strategy to reveal the mechanism of pinocembrin against ovarian cancer.

Ovarian cancer stands as the foremost cause of mortality among gynaecological diseases globally, characterized by high morbidity and mortality. Pinocembrin, a flavonoid from natural plant sources, exhibits diverse pharmacological properties. Despite its known pharmacological activities, its specific role in ovarian cancer treatment remains scarcely reported, and its precise molecular mechanism remains elusive. This study integrates network pharmacology and molecular docking techniques to explore pinocembrin's potential mechanism in ovarian cancer treatment. The targets of pinocembrin were compiled from the several online databases. Ovarian cancer targets were identified using the GeneCards database, with common target genes determined by data aggregation. Protein-protein interactions were analysed using the STRING platform. Subsequent Gene Ontology functional annotation and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed. Molecular docking assessed the binding affinity between potential targets and active compounds. Finally, target validity was verified through in vitro experiments. We identified 163 potential pinocembrin targets for ovarian cancer treatment. GO and KEGG analyses revealed pinocembrin's involvement in protein kinase activity, protein phosphorylation, protein kinase complexes and cancer pathways in ovarian cancer treatment. Molecular docking demonstrated strong binding affinity between pinocembrin and most potential target active sites. In vitro experiments suggested pinocembrin's potential to induce apoptosis in ovarian cancer cells through the AKT1-mTOR signalling pathway. This study comprehensively elucidates pinocembrin's potential targets and mechanisms against ovarian cancer, aiming to provide promising candidates for developing novel and effective alternative and/or complementary nutritional supplements for the clinical treatment of ovarian cancer.

Pharmacodynamic insights into maresin 1: Enhancing flap viability via the keap1/Nrf2 axis to control ROS-driven apoptosis and ferroptosis

Random flaps are widely used in tissue reconstruction, but the high incidence of flap necrosis after operation remains a significant challenge. Maresin 1 (MaR1), a mediator derived from docosahexaenoic acid, has been shown to have significant effects in resolving inflammation and promoting tissue regeneration. This study investigated the role of MaR1 in the survival of random flaps. Histological analysis, laser Doppler blood flow imaging, Masson trichrome staining, and survival area analysis were used to assess the viability of the flaps. Apoptosis, ferroptosis, oxidative stress, angiogenesis, and the underlying mechanisms were explored by examining the expression of specific molecules using immunofluorescence, western blotting, and other immunological and molecular biology techniques. The findings demonstrated that MaR1 could improve flap lifespan by significantly reducing oxidative stress, apoptosis, and ferroptosis, as well as by enhancing angiogenesis. The Keap1-Nrf2 pathway was upregulated by MaR1, which inhibited ROS-mediated apoptosis and ferroptosis. The protective effect of MaR1 on flap survival was abolished by ML385. Our findings indicate that MaR1 could be a novel therapeutic agent for enhancing flap treatment outcomes.

Enzymatic synthesis of ω-3 fatty acids-based structured phospholipids: A novel solvent-free preparation approach and its catalytic mechanism

ω-3 fatty acids-based phospholipids (ω-3-PLs) exhibit higher bioavailability within organisms and stability compared to polyunsaturated fatty acids in their neutral forms. In this study, ω-3 fatty acids enriched partial acylglycerols (ω-3-AGs) were produced from hydrolysis of algal oil firstly. The effects of hydrolysis processing parameters such as enzyme type, enzyme loading, oil-water ratio, reaction temperature, and time were evaluated. Subsequently, ω-3-PLs were prepared via interesterification of ω-3-AGs with PLs catalyzed by PLA1. When monoacylglycerols (MAGs) reached 60% in partial acylglycerols, the interesterification reaction showed a marked improvement, leading to the achievement of the optimal final ω-3-PLs content of 38.93%. Using molecular docking techniques to speculate on the catalytic mechanism, the results indicate that MAGs exhibit an affinity of up to −6.33 kcal/mol with PLA1, verifying their critical role in the interesterification process.

Mapping, Distribution, Function, and High-Throughput Methodological Strategies for Soil Microbial Communities in the Agroecosystem in the Last Decades

The intricate interplay between SMCs and agroecosystems has garnered substantial attention in recent decades due to its profound implications for agricultural productivity, ecosystem sustainability, and environmental health. Understanding the distribution of SMCs is complemented by investigations into their functional roles within agroecosystems. Soil microbes play pivotal roles in nutrient cycling, organic matter decomposition, disease suppression, and plant‒microbe interactions, profoundly influencing soil fertility, crop productivity, and ecosystem resilience. Elucidating the functional diversity and metabolic potential of SMCs is crucial for designing sustainable agricultural practices that harness the beneficial functions of soil microbes while minimizing detrimental impacts on ecosystem services. Various molecular techniques, such as next-generation sequencing and high-throughput sequencing, have facilitated the elucidation of microbial community structures and dynamics at different spatial scales. These efforts have revealed the influence of factors such as soil type, land management practices, climate, and land use change on microbial community composition and diversity. Advances in high-throughput methodological strategies have revolutionized our ability to characterize SMCs comprehensively and efficiently. These include amplicon sequencing, metagenomics, metatranscriptomics, and metaproteomics, which provide insights into microbial taxonomic composition, functional potential, gene expression, and protein profiles. The integration of multiomics approaches allows for a more holistic understanding of the complex interactions within SMCs and their responses to environmental perturbations. In conclusion, this review highlights the significant progress made in mapping, understanding the distribution, elucidating the functions, and employing high-throughput methodological strategies to study SMCs in agroecosystems.

Introducing palmfungi.org, an integrated fungal-host data platform

Palm fungi are a diverse and unique group mostly found on Arecaceae hosts. They have been studied for approximately 200 years resulting in a large number of known fungal species representing over 700 genera. The timeline of palm fungal studies could be roughly divided into three phases, based on the methods and frequency of reports. They are the “Historical palm fungi era”, “Classical palm fungi era” and “Molecular palm fungi era”. In the first two periods, the identification of palm fungi was based on morphology, which resulted in a considerable number of morphological species scattered across the data in books, monographs and papers. With the advancement of molecular techniques, studies on palm fungi accelerated. A large number of new species were introduced in the molecular era, especially from Asia, including China and Thailand. However, there is a necessity to link these three generations of studies into a single platform combining data related to host factors, geography and utilisation. Herein, we introduce the palm fungi website: https://palmfungi.org, an integrated data platform for interactive retrieval, based on palm and fungal species. This website is not only a portal for the latest, comprehensive species information on palm fungi, but also provides a new platform for fungal researchers to explore the host-specificity of palm fungi. Additionally, this study uses palmfungi.org and related data to briefly discuss the current status of research on the distribution of palm fungi populations, showing how palmfungi.org links fungi with their palm hosts. Furthermore, the website will act as a platform for collaboration amongst taxonomists, plant pathologists, botanists, ecologists and those who are interested in palms and their relationship with ecological sustainability.

B-208 Room Temperature Stable PCR Reagents Preparation Using Capillary-Mediated Vitrification Process

Abstract Background Polymerase chain reaction (PCR) is a sensitive, and most used molecular technique for the disease diagnosis. Reagents used for PCR is available as a one-step and ready-to-use PCR Master Mix, which contains Taq DNA polymerase, dNTPs, MgC12, PCR reaction buffer. However, to preserve bioactivity, PCR reagents must be stored and distributed as frozen. Desiccation of PCR reagents can avoid cold storage and improper handling of wet reagents and its associated errors. We discovered capillary-mediated vitrification (CMV) processes can stabilize different biomolecules and allow them to be stored at ambient temperature. Here, we hypothesized that the CMV process can preserve and improve PCR reagents thermal stability, which will reduce PCR processing time and improve the testing throughput. Methods The CMV process was performed by mixing PCR Master Mix individual or all reagents such as Taq DNA polymerase or Hot Start Taq DNA polymerase, dNTPs, 16S rRNA primers, MgC12, and PCR reaction buffer with an excipient buffer and adding the mixture to a porous scaffold. The loaded scaffolds were placed into our vitrification chamber and dried for 30 min. After drying, the CMV-stabilized sample was tested or packed in Mylar pouches and were stored at ambient and elevated temperatures for 42 days. On the day of testing, the stabilized samples were eluted with nuclease-free water and E. coli genomic DNA was added, and the 50 uL reaction was tested by conventional PCR. The PCR product was visualized under agarose gel electrophoresis. Results To assess whether the CMV process maintains PCR reagents activity and not interfering with the assay, the individual PCR reagents and as a Master mix are preserved. The CMV stabilized polymerase enzyme, primer, or dNTPs amplified DNA similar to frozen reagents and produced comparable PCR products. Notably, CMV stabilized PCR Master Mix which contains either Taq DNA polymerase or Hot Start Taq DNA polymerase enzyme, dNTPs, primers, MgC12, and reaction buffer yielded intact amplified PCR product, which is comparable to frozen reagents. Likewise, a high intense PCR product was observed in the stabilized PCR Master Mix reagents when stored at room and elevated temperatures for 42 days. Conclusions The study results indicate that the CMV process stabilized and improved the PCR Master Mix reagents activity. The study suggests that the CMV technology has the potential to enable ambient storage and shipping of PCR reagents for molecular diagnostic and other related applications.

Antimicrobial resistance and genotypic attributes of virulence among Vibrio spp. isolated from Japanese retail seafood

Bacterial diseases caused by Vibrio spp. are a significant impediment to the aquaculture industry owing to difficult-to-treat infections and mortality in farmed animals, affecting socioeconomic development. This study aimed to evaluate the role of Japanese retail seafood in the production of antimicrobial-resistant Vibrio spp. and pathogenic factors. One hundred seafood samples purchased from various retail supermarkets in Hiroshima, Japan, were examined using microbiological, phenotypic, and molecular techniques. One hundred and twenty-eight Vibrio isolates belonging to more than 11 species were identified and characterized. Among the 100 samples, 26 (26%) tested positive for Vibrio spp. with antimicrobial resistance, while 44 (44%) were positive for those carrying virulence determinants. Out of 128 isolates, V. alginolyticus was predominant (38%), followed by V. parahaemolyticus (29%), V. neocaledonicus (7.0%), V. cholerae (6.3%), among others. For virulence gene assessment, only six of the 10 virulence attributes, tlh (34.4%), VPI (29.7%), ompW (7.0%), toxR (5.5%), hlyA (3.9%), and ompU (3.1%), were detected in the isolates. tcpA, ctxA, ctxB, and tdh are absent. Eight V. cholerae isolates of international origin were VPI-positive, seven of which harbored the toxR regulon and other virulence genes. The blaTEM resistance gene was identified in V. alginolyticus (22 isolates). All 46 antimicrobial resistance gene-harboring isolates were phenotypically susceptible to cephalosporins, aztreonam, meropenem, tetracycline, chloramphenicol, and ciprofloxacin. Ampicillin resistance was the highest (91.3%), followed by colistin (30.4%) and fosfomycin (15.2%). The multiple-antibiotic resistance index ranged from 0.2‒0.47, suggesting that some isolates originate from high-risk contamination sources. Our results shed light on the incidence of potentially pathogenic Vibrio spp. and highlighted the importance of continuous monitoring to improve food safety in the seafood supply chain. These findings will be useful for developing microbiological risk assessments for retail seafood management.

Multiplex qPCR for the detection of bacteria associated with huanglongbing 'Candidatus Liberibacter asiaticus', 'Ca. L. americanus' and 16Sr IX group phytoplasma.

The occurrence of 'Candidatus Liberibacter' spp. and 'Ca. Phytoplasma' spp. associated with blotchy mottle symptoms poses challenges to huanglongbing (HLB) diagnosis using molecular techniques. The ability to detect multiple targets simultaneously and specifically is a key aspect met by qPCR. A set of primers and hydrolysis probes useful either in single or multiplex reactions for the detection and quantification of HLB-associated bacteria were developed. Sequences from conserved genes of the ribosomal proteins for Liberibacter and phytoplasma circumvent the lack of specificity and cross-reactivity problems related to 16S rDNA gene amplification, allowing precise and specific detection of HLB-associated bacteria in citrus and in the Liberibacter vector, Diaphorina citri. The triplex reaction exhibited high quality and precision as a robust tool for quantifying 'Ca. L. asiaticus' (CLas), 'Ca. L. americanus' (CLam) and 16SrIX phytoplasma. Triplex qPCR showed consistent results and comparable sensitivity to the RNR test, though Cq values were higher when compared to 16S rDNA qPCR. Detection tests using field samples indicate that the qPCR triplex can identify HLB-associated bacteria in samples with varying levels of symptoms, ranging from typical to asymptomatic. Assessment of field samples from growers indicated more than 78.6% had Cq lower than 35.0, below the cut-off established for qPCR reactions used in this work. qPCR triplex is a safe, specific, and sufficiently sensitive technique for detecting CLas, CLam and 16Sr IX phytoplasma simultaneously, in both citrus and D. citri samples. Its application is of importance in assisting growers in making decisions for HLB management.

Methods used in sea turtle genetic and molecular studies

Sea turtles have a tropical and subtropical distribution and can be found in nearly all seas and oceans. They have been the subject of considerable genetic research. However, it does not yet appear that the molecular techniques used for these genetic studies follow a consensus or universal set of tools to be followed for subsequent studies. This is not desirable since it may preclude data exchange and use among studies. Thus, the aim of this review was to survey the main genetic and molecular methods used for sea turtle research worldwide. To achieve this goal, a total of 95 scientific papers were compiled from online databases. We considered articles that used molecular tools for genetic analysis and provided detailed locality data. The following aspects were assessed: species studied, local of sample collection, type of tissue used for molecular studies and type of genetic material used. The seven known sea turtle species have different distribution patterns, with some overlapping of occurrence. Chelonia mydas has been studied genetically along the coasts of all continents. Skin is the most common type of tissue used for molecular analyses. From genetic studies on sea turtles, it is possible to verify the occurrence of hybridism. This phenomenon is relevant to the conservation of the species and was reported in six articles on this topic. Therefore, it is considered that genetic and molecular assays in sea turtles are important tools for biological evaluation and protection. The aim was to survey the main methods used in genetic and molecular research on marine turtles. Keywords: DNA, Chelonian, Testudines, marine turtles

A pilot study on the epidemiology, diagnosis and characterization of Echinococcus granulosus sensu lato in sheep, goats and dogs in Siirt province of Türkiye revealed remarkable adaptation of Echinococcus canadensis (G6/G7) in goats

Cystic echinococcosis (CE), caused by Echinococcus granulosus sensu lato (s.l.), represents one of the most significant zoonotic diseases globally, affecting both humans and animals. The objective of this study was to ascertain the prevalence of E. granulosus sensu lato in sheep and goats in a pilot region with a one-year slaughterhouse follow-up period and to determine the genetic differences and haplotypes among sheep, goat, and dog isolates. To this end, the prevalence of CE cysts was determined by monitoring the slaughter of sheep and goats at least three days a week at a slaughterhouse in the Siirt province of Türkiye during 2023. Additionally, faecal samples were collected from stray dogs and analysed using both flotation and molecular techniques. The presence of CE cysts was identified in 569 (11.12 %) of the 5119 sheep and 66 (2.31 %) of the 2860 goats after slaughtering. The highest positivity was observed in November (20.39 %), while the lowest was recorded in July (5.62 %). Of the sheep that detected positive, 25 (4.39 %) were less than one year old, while 544 (95.61 %) were older than one year. Of the infected sheep, 26 (4.57 %) were male and 543 (95.43 %) were female. 204 (35.85 %) sheep exhibited fluid-filled CE cysts, 338 (59.40 %) displayed calcification, and 27 (4.75 %) demonstrated the presence of newly developed cysts. The highest positivity was observed in December (5.83 %), while the lowest was recorded in May (0.62 %) in goats. Of the positive goats, two (3 %) were less than one year old, while the remaining 64 (97 %) were older than one year. Of the goats infected with CE cysts, 10 (15.15 %) were male and 56 (84.85 %) were female. Of the cysts, 56.1 % were fluid-filled, 42.4 % were calcified and 1.5 % were newly developed. Following DNA sequence analysis of CE cyst isolates obtained from the slaughterhouse, all 61 sheep sequences were identified as E. granulosus s.s. (G1/G3). Of the 13 goat isolates, seven were identified as E. granulosus s.s. (G1/G3), while the remaining six were classified as E. canadensis (G6/G7). The centrifugal flotation method was employed to detect the presence of Isospora spp. oocysts in eight dogs, Toxocara canis and hookworm eggs in three dogs each, and Dipyllidium caninum eggs in one dog. A total of 54 dog faeces were examined. No Taeniid eggs were observed in any of the dogs. Following PCR analysis of the mt-CO1 gene region in the dog faecal samples, four samples were positive for a 875 bp band. Only one of these bands was suitable for sequence analysis, which confirmed it as E. granulosus s.s. (G1/G3).

Elevated proline and melatonin alter the methylation pattern in the promoter of their biosynthesis genes in rice

Salt stress upregulates osmoprotectants proline and melatonin in plants, and their exogenous application under salt stress can protect them from stress. While melatonin-induced plant epigenetic reprogramming is known in plants, such a mechanism is yet to be identified in the case of proline. The studies conducted so far used proline and melatonin along with one or more stress factors to look into their stress-alleviating effects. We investigated the impact of externally applied proline and melatonin on the methylation of promoter sequences of proline and melatonin biosynthesis genes in 14-day-old rice plants using a combination of biochemical, bioinformatics, and molecular techniques. Our findings demonstrate that exogenously applied proline and melatonin elevate endogenous levels of these compounds in rice, mimicking stress conditions in plants, as the biochemical assays corroborated the activation of antioxidant systems, particularly ascorbate peroxidase (APX) and catalase (CAT). Bioinformatics analysis unveiled multiple stress-responsive transcription factor binding sites on gene promoters associated with proline and melatonin biosynthesis pathways. Restriction analysis using the isoschizomer pair MspI/HpaII revealed distinct cytosine methylations at the restriction sites on the promoters analyzed in plants treated with proline and melatonin compared to the control. Differential methylation identified in the promoter sequences were matching with the biosynthesis of proline and melatonin and also the antioxidant enzyme levels. These observations are consistent with previous transcriptome data of proline and melatonin biosynthesis genes, providing insights into underlying regulatory mechanisms of proline and melatonin biosynthesis, their role in epigenome control during abiotic stress, and the evolution of various stress-tolerant varieties.

ATR-FTIR spectroscopy to evaluate serum protein expression in a murine cerebral ischemia model

Stroke is a prevalent vascular disease that causes disability and death worldwide. Molecular techniques have been developed to assess serum concentrations of biomarkers associated with this disease, such as some proteins. ATR-FTIR was proposed as an alternative technique to determine protein expression during the early stages of stroke. Serum samples from sham, ischemic, and ischemic treated with estradiol benzoate (EB; as a neuroprotective agent) male rats were evaluated at 0, 2-, 4-, 6-, 12-, and 24-hours post-ischemia. The analysis was developed in the mid-infrared region but mainly focused on the protein region (1500 to 1700 cm−1), where it was possible to observe the modulation in the absorbance intensity. The peaks at 1545, 1645, 1635, and 1650 cm−1 associated with amide II, amide I, β −sheets, and α-helixes, respectively, were prominent peaks where protein modulation was observed. The results demonstrate that infrared spectroscopy could be a good alternative technique to determine the modulation of protein expression during stroke events.

Evil and allies: Opportunistic gulls as both spreaders and sentinels of antibiotic‐resistant bacteria in human‐transformed landscapes

Abstract Human‐transformed residuals, especially those derived from human waste (dumps), farmland, and livestock are involved in the emergence of antibiotic‐resistant bacteria (ARB) in the environment. Wildlife can act as vectors of ARB dispersal through different environments, but also as sentinels to detect the early spread and determine ARB sources. The development of integrated monitoring programmes focused on wildlife would help to anticipate the risks of ARB to humans and livestock. We used the yellow‐legged gull (Larus michahellis) as a model species to investigate and monitor the spatial patterns of ARB dispersal across an extensive farmland region located in northeastern Spain (Lleida). By integrating GPS tracking data and ARB clinical testing for 26 individuals within a network analysis framework, we modelled the risk of spatial pathogen spread through faeces during the bacteria‐transmission latency period (16 days after sample collection). Additionally, we created a connectivity network to determine the main sources of ARB in the area, focusing on three main habitats of special risk for infection: dumps, livestock facilities, and irrigation ponds. Seven individuals were infected by Escherichia coli, with one also co‐infected with Listeria monocytogenes and Salmonella spp. Potential pathogen dispersal distances ranged from 1.13 km to 23.13 km from the breeding colony. Our network analyses revealed 54 main nodes (i.e. high‐risk habitats recurrently visited by tracked gulls) and 1182 links among them. Our findings revealed a high degree of connectivity between the breeding area, located in a shallow lake, and nearby dumps, highlighting them as significant contributors to ARB dispersal. Synthesis and applications: The integration of GPS data, pathogen testing and network analyses can shed further light on pathogen dynamics by creating spatial risk maps and identifying ARB sources. In combination with complementary molecular epidemiology techniques within a One Health framework, our approach can emerge as an important tool for monitoring ARB dynamics within highly human‐transformed ecosystems. This may empower managers for the development of targeted ARB monitoring programmes and effective mitigation strategies, ultimately improving both animal and public health.

In-silico Investigation of Ginseng Phytoconstituents as Novel Therapeutics Against MAO-A.

Ginseng (Panax ginseng) is a herb of medicinal and nutritional importance. Ginseng has been used since ancient times for the treatment of numerous ailments as it has many therapeutic properties. Several phytoconstituents are present in Panax ginseng that possess a variety of beneficial pharmacological properties. To explore the potential of phytoconstituents of Panax ginseng in the treatment of depression, a molecular modeling technique was utilized targeting monoamine oxidase-A (MAO-A). A total of sixty-one phytoconstituents of ginseng were drawn with the help of ChemBioDraw Ultra 12.0 software and PDBs for MAO-A enzyme were retrieved from the RCSB PDB database. The prepared ligands were screened for MAO-A properties using the software Molegro Virtual Docker (MVD 2010.4.1.0). All the prepared ligands were evaluated for drug-likeliness properties using Swiss ADME. Among the docking studies of 60 Ginseng phytochemicals including one standard, 15 phytoconstituents with the highest dock score and better binding interactions were selected further for absorption, distribution, metabolism and excretion (ADME) studies. Stachyose (-227.287, 17 interactions), Raffinose (-222.157, 14 interactions), and Ginsenoside Rg1 (-216.593, 10 interactions) were found to possess better interactions as compared to Clorgyline taken as a standard drug. Stachyose was found to be the most potent inhibitor of MAO-A enzyme under investigation and can be a potential lead molecule for the development of newer phytochemical-based treatment of depression.

Investigation of the molecular interaction between apraclonidine, an α2-adrenergic receptor agonist, and bovine serum albumin using fluorescence and molecular docking techniques

Apraclonidine (APR) is a potent and selective α2-adrenergic receptor agonist used in the diagnosis of Horner’s Syndrome, and the residuals of APR that accumulate in tissues of animals can cause central nervous and cardiovascular systems influences in humans. Therefore, to understand the influence of APR on human health, we examined the interaction of APR with the carrier protein in plasma, bovine serum albumin (BSA). The BSA fluorescence signal was quenched due to the APU–BSA complex formation and a weak binding affinity was estimated between APR and BSA. The inclusion of fluorescence, UV–vis absorption, molecular docking, and dynamics simulation techniques employed to broadly investigate the combination of APR with BSA at typical physiological conditions. The thermodynamic results revealed that enthalpy (ΔH0) and entropy (ΔS0) changes were computed as +11.14 kJ mol−1 and +97.56 J mol−1 K−1, respectively, which represented the binding is principally entropy-driven and the hydrophobic forces acting a significant role in the reaction. Analysis of synchronous and 3-D fluorescence signals revealed microenvironmental variations close to BSA’s Trp and Tyr residues upon APR addition. Both the competitive site marker as well as molecular docking results detected that APR exhibited a stronger binding affinity towards Drug Site 2 (DS2) compared to Drug Site 1 (DS1).

Effect of Cu2+ on the binding of catechins to zein through multi-spectral and in silico analyses

This study aimed to elucidate how Cu2+ influences the interaction between catechins and zein using multi-spectral and molecular modeling techniques. As a result, UV–vis spectra revealed characteristic changes, indicating chelation between catechins and Cu2+ at a 1:1 M ratio. Fluorescence spectra further confirmed interaction through a static quenching mechanism between catechins/catechin-Cu2+ complexes and zein. Catechins induced changes in the microenvironment and hydrophobicity surrounding the binding site of zein, whereas Cu2+ had minimal impact on these aspects. CD spectra underscored catechins' role in altering zein's secondary structure conformation, alongside Cu2+. Various types of interactions (hydrophobic, hydrogen bonding, electrostatic, and van der Waals) contributed to the binding of catechins/catechin-Cu2+ complexes with zein. Molecular modeling elucidated key residues and binding conformations, highlighting the significance of hydrophobic interactions and hydrogen bonding in their association. These findings not only deepen our understanding of catechin-Cu2+-zein interactions but also underscore their potential applications in the food industry.

Antioxidant, Xanthine oxidase (XO) Inhibitory, Hypouricemic Effect Evaluation and GCMS Analysis of Ethanolic Extract of Piper chaba Stem: Supported by in vitro, in vivo, and molecular docking experiments

BackgroundPiper chaba has been traditionally using for its potential health benefits. The principal goal of this investigation was to assess the pharmacological characteristics of the EEPCS, focusing on investigating its potential antihyperuricemic effects, ability to inhibit xanthine oxidase (XO) enzyme, and antioxidant properties. MethodsThe antioxidant capability of the extract was assessed using the DPPH radical assay. The extract was evaluated in vitro aimed at its xanthine oxidase inhibitory (XOI) action and further experimented for its antihyperuricemic effect on the Potassium oxonate (PO) induced hyperuricemia murine model. Besides, molecular docking techniques were employed to interpret the inhibitory mechanism of the compounds identified through GCMS analysis of the EEPCS. ResultsThere was no safety concern at doses up to 3000 mg/kg of the extract as confirmed by mice model studies. The extract displayed potent the antioxidant properties during quantitative antioxidant test (IC50=104.9 ± 0.99 µg/mL). In XOI test, the extract was found to be effective with an IC50 value of 26.14 μg/mL. In addition, the extract was found to decrease the Serum uric acid (SUA) levels by 36.72 and 61.84% at 100 and 200 mg/kg body weight respectively, compared to the hyperurecaemic animals. The GCMS analysis of the extract revealed eighteen distinct compounds, which were docked against human peroxiredoxin 5 and xanthin oxidase proteins. Selina-3,7(11)-diene (CID: 6429221) was the most effective phytochemical for the human peroxiredoxin 5 protein having binding score -6.2 Kcal/mol compared to standard ascorbic acid (5.6 Kcal/mol). Besides, gamma-muurolene (-7.9 Kcal/mol) was the most effective phytochemical for the XO protein compared to standard allopurinol (-6.4 Kcal/mol). ConclusionOur experimental data showed that the EEPCS has potentiality as an antioxidant and hypouricemic agent that could be contributed to treat and manage oxidative stress (OS), gout and hyperuricemia.

UPLC-Q-TOF-MS/MS Combined with Network Pharmacology, Molecular Docking, and Animal Verification Reveals the Mechanism of Insomnia Treatment by Shen Qi Wu Wei Zi Capsules.

Shen Qi Wu Wei Zi capsules (SQWWZ) are often used to treat insomnia; however, the potential therapeutic mechanism is still unclear. This study aimed to investigate the mechanism underlying the therapeutic effects of the Shen Qi Wu Wei Zi capsules on insomnia. The components of SQWWZ were identified using the UPLC-Q-TOF-MS/MS technique in conjunction with relevant literature. Insomnia-related targets were searched in the Gene- Cards and DisGeNET databases, and the intersection targets were obtained using a Venn diagram. A component-target-insomnia network diagram was constructed using Cytoscape 3.7.2 software. Core targets underwent GO and KEGG enrichment analyses. Molecular docking techniques were employed to verify the key proteins involved in the pathway and their corresponding compounds. Insomnia was induced in SD rats through the intraperitoneal injection of pchlorophenylalanine (DL-4-chlorophenylalanine, PCPA). The rats were treated orally with SQWWZ, and the serum levels of 5-HT and GABA in each group were determined using ELISA. Histological analysis of hippocampal tissue sections from the rats was performed using HE staining. Using UPLC-Q-TOF-MS/MS and reviewing relevant literature, we identified 49 components of SQWWZ. Additionally, we obtained 1,043 drug targets and 367 insomnia-related targets. Among these, 82 targets were found to be common to both drug and insomnia targets. Following drug administration, rats in the treatment group exhibited a significant increase in the serum levels of 5-HT and GABA. Moreover, histological analysis using HE staining revealed neatly arranged hippocampal neuronal cells in the treated rats. The active components of SQWWZ had good inhibition of insomnia. This study provides a reference and guidance for the in-depth study of SQWWZ for the treatment of insomnia.

Quercetin restores respiratory mucosal barrier dysfunction in Mycoplasma gallisepticum-infected chicks by enhancing Th2 immune response.

Mycoplasma gallisepticum (MG) has long been a pathogenic microorganism threatening the global poultry industry. Previous studies have demonstrated that the mechanism by which quercetin (QUE) inhibits the colonization of MG in chicks differs from that of antibiotics. However, the molecular mechanism by which QUE facilitates the clearance of MG remains unclear. The aim of this study was to investigate the molecular mechanism of MG clearance by QUE, with the expectation of providing new options for the treatment of MG. A model of MG infection in chicks and MG-induced M1 polarization in HD-11 cells were established. The mechanism of QUE clearance of MG was investigated by evaluating the relationship between tracheal mucosal barrier integrity, antibody levels, Th1/Th2 immune balance and macrophage metabolism and M1/M2 polarization balance. Furthermore, network pharmacology and molecular docking techniques were employed to explore the potential molecular pathways connecting QUE, M2 polarization, and fatty acid oxidation (FAO). The findings indicate that QUE remodels tracheal mucosal barrier function by regulating tight junctions and secretory immunoglobulin A (sIgA) expression levels. This process entails the regulatory function of QUE on the Th1/Th2 immune imbalance that is induced by MG infection in the tracheal mucosa. Moreover, QUE intervention impeded the M1 polarization of HD-11 cells induced by MG infection, while simultaneously promoting M2 polarization through the induction of FAO. Conversely, inhibitors of the FAO pathway impede this effect. The results of computer network analysis suggest that QUE may induce FAO via the PI3K/AKT pathway to promote M2 polarization. Notably, inhibition of the PI3K/AKT pathway was found to effectively inhibit M2 polarization in HD-11 cells, while having a limited effect on FAO. QUE promotes M2 polarization of HD-11 cells to enhance Th2 immune response through FAO and PI3K/AKT pathways, thereby restoring tracheal mucosal barrier function and ultimately inhibiting MG colonization.