Characterization of NLRP3 inflammasome components in the endangered Chinese giant salamander (Andrias davidianus).

Chinese giant salamander (Andrias davidianus) is the largest extant urodela species and has unique evolutionary position. Studying the immune system of Chinese giant salamander contributes to understanding the evolution of immune systems of vertebrates. The NLR-related protein 3 (NLRP3) inflammasome comprised of NLRP3, ASC and caspase-1 play important roles in the host innate immunity. However, little is know about the NLRP3 inflammasome components in Chinese giant salamander. In this study, the NLRP3, apoptosis-associated speck-like protein (ASC) and caspase-1 (adaNLRP3, adaASC and adaCaspase-1) were characterized from Chinese giant salamander. The proteins of these three genes shared similar motifs and structures with their mammalian counterparts, with a PYD motif, a nucleotide-binding domain (NACHT) motif, and four leucine-rich repeat domain (LRR) motifs identified in adaNLRP3, a pyrin domain (PYD) motif and a caspase recruitment domain (CARD) motif in adaASC, and a CARD motif and a CASc motif in adaCaspase-1. These three genes were constitutively expressed in the skin, heart, lung, kidney, muscle, brain, spleen, and liver of Chinese giant salamander. Following Aeromonas hydrophia infection, all the three genes were up-regulated in various tissues. Molecular docking analysis revealed that the key residues involved in forming the adaNLRP3/adaASC complex were located in the PYD motifs, and that involved in forming the adaASC/adaCaspase-1 complex were located in the CARD motifs. Further analysis revealed that the hydrogen bonds and salt bridges had crucial roles in the formation of adaNLRP3/acaASC and adaASC/adaCaspase-1 complexes. To the best of our knowledge, this is the first report on the NLRP3 inflammasome components in Chinese giant salamander which will be helpful in further understanding the function of the NLRP3 inflammasome and in elucidating its role in the immune response to microbes.

Spastin regulates ER-mitochondrial contact sites and mitochondrial homeostasis.

Mitochondria-endoplasmic reticulum (ER) contact sites (MERCs) emerged to play critical roles in numerous cellular processes, and their dysregulation has been associated to neurodegenerative disorders. Mutations in the SPG4 gene coding for spastin are among the main causes of hereditary spastic paraplegia (HSP). Spastin binds and severs microtubules, and the long isoform of this protein, namely M1, spans the outer leaflet of ER membrane where it interacts with other ER-HSP proteins. Here, we showed that overexpressed M1 spastin localizes in ER-mitochondria intersections and that endogenous spastin accumulates in MERCs. We demonstrated in different cellular models that downregulation of spastin enhances the number of MERCs, alters mitochondrial morphology, and impairs ER and mitochondrial calcium homeostasis. These effects are associated with reduced mitochondrial membrane potential, oxygen species levels, and oxidative metabolism. These findings extend our knowledge on the role of spastin in the ER and suggest MERCs deregulation as potential causes of SPG4-HSP disease.

Etomoxir repurposed as a promiscuous fatty acid mimetic chemoproteomic probe.

Etomoxir has been used for decades as a popular small molecule inhibitor of carnitine palmitoyltransferase I, Cpt1, to block mitochondrial fatty acid ß-oxidation. To test the specificity of etomoxir, we generated click chemistry-enabled reagents to label etomoxir binding proteins in situ. Etomoxir bound to Cpt1, but also bound to a large array of diverse proteins that metabolize and transport fatty acids in the cytoplasm, peroxisome, and mitochondria. Many of the most abundant proteins identified in primary hepatocytes were peroxisomal proteins. The loss of Pex5, required for the import of peroxisomal matrix proteins, eliminated many of these etomoxir-labeled proteins. By utilizing the promiscuous, covalent, and fatty acid mimetic properties of etomoxir, etomoxir targets of fatty acid ω-oxidation were revealed following the loss of Pex5. These data demonstrate that etomoxir is not specific for Cpt1 and is not appropriate as a tool to distinguish the biological effects of fatty acid oxidation.

Metabolomics-based profiling of anti-inflammatory compounds from Mentha spicata in shanghe, China.

Mentha spicata is a popular herb used in foods, cosmetics, and medicines. In the present study, liquid chromatography-mass spectrometry-based metabolomics analysis and the zebrafish model were used to investigate the potential biomarkers of M. spicata growing in Shanghe County (Shandong Province, China) and their anti-inflammatory properties. Network pharmacology and molecular docking were performed to screen the main targets of the characteristic compounds to understand their mechanisms of action. Nine potential markers including sugars (1,2), polyphenolic acids (3-5), and flavonoids (6-9) were identified from the species. The inhibitory effects on leukocyte migration confirmed that compounds 1 and 3-9 played a positive role in the protective effect of Shanghe M. spicata (SM) extract against inflammation. Akt (protein kinase B), EGFR (epidermal growth factor receptor), and MMP9 (matrix metalloproteinase 9) were the core target proteins of the identified compounds in the anti-inflammatory process. The most significant Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment terms were response to abiotic stimulus (Biological Process), carbohydrate derivative binding (Molecular Function), and pathways in cancer. In docking simulations, 3-p-coumaroylquinic acid (3-PC, 4) and cirsimaritin (CN, 7) exhibited the highest potential affinity to the active sites of Akt and EGFR proteins, respectively; additionally, 5-demethylsinensetin (5-DS, 9) and luteolin (LN, 6) were considered the most suitable ligands for the MMP9 protein. The present study highlighted the use of SM resources as functional products with health benefits.

The molecular mechanisms underlying optical isomer arbutin permeating the skin: The molecular interaction between arbutin and skin components.

Arbutin, a typical optical isomer, has garnered widespread acclaim in the whitening cosmetics for its favorable efficacy and safety. However, the molecular mechanisms underlying α-arbutin and ß-arbutin permeating across the skin have not elucidated clearly yet. Herein we aimed to unveil how α-arbutin and ß-arbutin interacted with keratin or SC lipids, further demonstrating their relationship with their drug permeability. We found that α-arbutin displayed significantly higher drug accumulation into the porcine skin than ß-arbutin within 24 h through in vitro permeation test. Moreover, α-arbutin predominantly induced the alternations of secondary structure of amide II during the drug permeation, which was favorable for α-arbutin permeation. On the contrary, ß-arbutin exhibited an observable effect on the stretching vibration of SC lipids, possessing a significantly stronger mixing energy, binding energy and compatibility with ceramide (Cer) than that of α-arbutin, which ultimately restricted its permeation. Interestingly, free fatty acids and ceramides of the SC lipids specifically utilized its oxygen atom of carboxyl group to dock the arbutin molecules, enhancing their affinity with ß-arbutin, as confirmed by molecular simulation and 13Carbon Nuclear Magnetic Resonance. Nevertheless, a favorable compatibility between α-arbutin and keratin was observed. It was emphasized that the distinct spatial configuration and opposite optical rotation of arbutin was the leading factor impacting the intermolecular force between arbutin and the SC, and resulted in a diverse drug permeation. In cellular and in vivo skin pharmacokinetic studies, α-arbutin also possessed a higher cellular uptake and topical bioavailability than ß-arbutin. This study revealed the transdermal permeation mechanisms of optical isomer arbutin at the molecular levels, providing methodological reference for the investigations of permeation behaviors of other isomers with similar spatial configuration.

Small molecule inhibitor binds to NOD-like receptor family pyrin domain containing 3 and prevents inflammasome activation.

Despite recent advances in the mechanism of oxidized DNA activating NLRP3, the molecular mechanism and consequence of oxidized DNA associating with NLRP3 remains unknown. Cytosolic NLRP3 binds oxidized DNA which has been released from the mitochondria, which subsequently triggers inflammasome activation. Human glycosylase (hOGG1) repairs oxidized DNA damage which inhibits inflammasome activation. The fold of NLRP3 pyrin domain contains amino acids and a protein fold similar to hOGG1. Amino acids that enable hOGG1 to bind and cleave oxidized DNA are conserved in NLRP3. We found NLRP3 could bind and cleave oxidized guanine within mitochondrial DNA. The binding of oxidized DNA to NLRP3 was prevented by small molecule drugs which also inhibit hOGG1. These same drugs also inhibited inflammasome activation. Elucidating this mechanism will enable the design of drug memetics that treat inflammasome pathologies, illustrated herein by NLRP3 pyrin domain inhibitors which suppressed interleukin-1ß (IL-1ß) production in macrophages.

SPI1-KLF1/LYL1 axis regulates lineage commitment during endothelial-to-hematopoietic transition from human pluripotent stem cells.

PU.1 (SPI1) is pivotal in hematopoiesis, yet its role in human endothelial-to-hematopoietic transition (EHT) remains unclear. Comparing human in vivo and in vitro EHT transcriptomes revealed SPI1's regulatory role. Knocking down SPI1 during in vitro EHT led to a decrease in the generation of hematopoietic progenitor cells (HPCs) and their differentiation potential. Through multi-omic analysis, we identified KLF1 and LYL1 - transcription factors specific to erythroid/myeloid and lymphoid cells, respectively - as downstream targets of SPI1. Overexpressing KLF1 or LYL1 partially rescues the SPI1 knockdown-induced reduction in HPC formation. Specifically, KLF1 overexpression restores myeloid lineage potential, while LYL1 overexpression re-establishes lymphoid lineage potential. We also observed a SPI1-LYL1 axis in the regulatory network in in vivo EHT. Taken together, our findings shed new light on the role of SPI1 in regulating lineage commitment during EHT, potentially contributing to the heterogeneity of hematopoietic stem cells (HSCs).

Enhancement of the gelling properties of Aristichthys nobilis: Insights into intermolecular interactions between okra polysaccharide and myofibrillar protein.

The effects of various contents of okra polysaccharide (OP) (0%-1%) on myofibrillar protein (MP) gelation and the interaction mechanism between OP and MP were investigated. OP improved the gelling properties of MP with an additive limitation of 0.75%. Rheological analysis demonstrated that the addition of OP enhanced the interactions between MPs, resulting in a denser intermolecular gel network structure. The addition of OP shifted the I850/I830 of Fourier transform infrared spectroscopy, indicating that hydrogen bonds were formed between OP and MP. Adding OP promoted the transition from α-helix to ß-sheet in the MP. OP exposed the hydrophobic groups of MPs and increased the number of hydrophobic interactions between them, favoring the formation of a dense gel network. Molecular docking predicted that hydrogen bonds were the main force involved in the binding of OP and MP. Moderate OP promoted the aggregation of MPs and improved their functional properties, facilitating heat-induced gelation.

Mutation in the Kinase Domain Alters the VEGFR2 Membrane Dynamics.

BACKGROUND: Recently, the substitution R1051Q in VEGFR2 has been described as a cancer-associated "gain of function" mutation. VEGFR2R1051Q phosphorylation is ligand-independent and enhances the activation of intracellular pathways and cell growth both in vitro and in vivo. In cancer, this mutation is found in heterozygosity, suggesting that an interaction between VEGFR2R1051Q and VEGFR2WT may occur and could explain, at least in part, how VEGFR2R1051Q acts to promote VEGFR2 signaling. Despite this, the biochemical/biophysical mechanism of the activation of VEGFR2R1051Q remains poorly understood. On these bases, the aim of our study is to address how VEGFR2R1051Q influences the biophysical behavior (dimerization and membrane dynamics) of the co-expressed VEGFR2WT. METHODS: We employed quantitative FLIM/FRET and FRAP imaging techniques using CHO cells co-transfected with the two forms of VEGFR2 to mimic heterozygosity. RESULTS: Membrane protein biotinylation reveals that VEGFR2WT is more exposed on the cell membrane with respect to VEGFR2R1051Q. The imaging analyses show the ability of VEGFR2WT to form heterodimers with VEGFR2R1051Q and this interaction alters its membrane dynamics. Indeed, when the co-expression of VEGFR2WT/VEGFR2R1051Q occurs, VEGFR2WT shows reduced lateral motility and a minor pool of mobile fraction. CONCLUSIONS: This study demonstrates that active VEGFR2R1051Q can affect the membrane behavior of the VEGFR2WT.

Larvicidal activity of ß-Citral: An In-vitro and In-silico study to understand its potential against mosquito.

Tropical and subtropical regions face millions of deaths from mosquito-borne illnesses yearly. Insecticides prevent transmission but pose health risks like dermatitis and allergies. The primary objective was to mitigate the recurring dependence on synthetic insecticides, thereby curbing the development of mosquito resistance. Leaves of Cymbopogon flexuosus (lemongrass) was collected from Mayurbhanj, India, processed, then extracted by steam distillation for essential oils & analyzed spectroscopically. Larvicidal assays were performed across varying concentrations, revealing the significant mortality induced by the Cymbopogon flexuosus extract against Anopheles stephensi larvae. 3D structure was modelled by using G protein-coupled receptors (GPCR) sequence and structural stability was also validated. After docking the binding free energy was determined from GPCR protein with ß-citral complex. Molecular dynamics (MD) study was conducted on the docked pose that displayed an optimal interactome profile. The larvicidal assay at the 12th and 24th hour revealed the highest LC50 (lethal concentration) of 23.493 ppm and 19.664 ppm . ß-Citral has a high binding affinity and an identifiable binding site, which suggests that it may play a larvicidal role in regulating the receptor's function by creating stable complexes with it. ß-Citral from lemongrass oils has potential larvicidal activity and effective against GPCR family 1 of mosquito and highly effective repellents against mosquito-borne diseases.

Caffeine weakens the astringency of epigallocatechin gallate by inhibiting its interaction with salivary proteins.

The astringency of green tea is an integrated result of the synergic and antagonistic effects of individual tea components, whose mechanism is highly complex and not completely understood. Herein, we used an epigallocatechin gallate (EGCG)/caffeine (CAF)/saliva model to simulate the oral conditions during tea drinking. The effect of CAF on the interaction between EGCG and salivary proteins was first investigated using molecular docking and isothermal titration calorimetry (ITC). Then, the rheological properties and the micro-network structure of saliva were studied to relate the molecular interactions and perceived astringency. The results revealed that CAF partially occupied the binding sites of EGCG to salivary proteins, inhibiting their interaction and causing changes in the elastic network structure of the salivary film, thereby reducing astringency.

Assessing the influences of ß-glucan on highland barley starch: Insights into gelatinization and molecular interactions.

Developing highland barley products is complex, possibly due to the presence of ß-glucan in highland barley. This study aims to investigate the impact of ß-glucan on the physicochemical properties, microstructure, and molecular interactions of highland barley starch (HBS) during gelatinization and aging. Increasing the ß-glucan content significantly reduced peak viscosity, setback viscosity, and breakdown viscosity, indicating altered gelatinization behavior. The ß-glucan content increase caused a significant drop in peak viscosity. With 20% ß-glucan addition, it reduced by 883 mPa·s, nearly 38%. Rheological analysis showed a transition from a solid-like to a liquid-like texture or quality, ultimately leading to a shear-thinning behavior. Fourier-transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD) confirmed the interaction between HBS and ß-glucan via intermolecular hydrogen bonding, promoting the formation of double helical structures in starch. These findings provide a deeper understanding of the role of ß-glucan in the processing of highland barley, highlighting its influence on the starch's properties.

Cytochrome c prompts the recruitment of its nuclear partners SET/TAF-Iß and NPM1 into biomolecular condensates.

Compartmentalization of proteins by liquid-liquid phase separation (LLPS) is used by cells to control biochemical reactions spatially and temporally. Among them, the recruitment of proteins to DNA foci and nucleolar trafficking occur by biomolecular condensation. Within this frame, the oncoprotein SET/TAF-Iß plays a key role in both chromatin remodeling and DNA damage response, as does nucleophosmin (NPM1) which indeed participates in nucleolar ribosome synthesis. Whereas phase separation by NPM1 is widely characterized, little is known about that undergone by SET/TAF-Iß. Here, we show that SET/TAF-Iß experiences phase separation together with respiratory cytochrome c (Cc), which translocates to the nucleus upon DNA damage. Here we report the molecular mechanisms governing Cc-induced phase separation of SET/TAF-Iß and NPM1, where two lysine-rich clusters of Cc are essential to recognize molecular surfaces on both partners in a specific and coordinated manner. Cc thus emerges as a small, globular protein with sequence-encoded heterotypic phase-separation properties.

Incorporation of cross-linked/acetylated tapioca starches on the gelling properties, rheological behaviour, and microstructure of low-salt myofibrillar protein gels: Perspective on phase transition.

This study investigated the gelling properties, rheological behaviour, and microstructure of heat-induced, low-salt myofibrillar protein (MP) gels containing different levels (2%, 4%, 6%, and 8%, w/w) of cross-linked (CTS) or acetylated (ATS) tapioca starch. The results indicated that either CTS or ATS significantly enhanced the gel strength and water-holding capacity of low-salt MP gels (P < 0.05), an outcome verified by the rheological behaviour test results under different modes. Furthermore, iodine-staining images indicated that the MP-dominated continuous phase gradually transited to a starch-dominated phase with increasing CTS or ATS levels, and 4% was the critical point for this phase transition. In addition, hydrophobic interactions and disulphide bonds constituted the major intermolecular forces of low-salt MP gels, effectively promoting phase transition. In brief, modified tapioca starches possess considerable potential application value in low-salt meat products.

Exo1 cooperates with Tel1/ATM in promoting recombination events at DNA replication forks.

Tel1/ataxia telangiectasia mutated (ATM) kinase plays multiple functions in response to DNA damage, promoting checkpoint-mediated cell-cycle arrest and repair of broken DNA. In addition, Saccharomyces cerevisiae Tel1 stabilizes replication forks that arrest upon the treatment with the topoisomerase poison camptothecin (CPT). We discover that inactivation of the Exo1 nuclease exacerbates the sensitivity of Tel1-deficient cells to CPT and other agents that hamper DNA replication. Furthermore, cells lacking both Exo1 and Tel1 activities exhibit sustained checkpoint activation in the presence of CPT, indicating that Tel1 and Exo1 limit the activation of a Mec1-dependent checkpoint. The absence of Tel1 or its kinase activity enhances recombination between inverted DNA repeats induced by replication fork blockage in an Exo1-dependent manner. Thus, we propose that Exo1 processes intermediates arising at stalled forks in tel1 mutants to promote DNA replication recovery and cell survival.

Regulation of the DLC3 tumor suppressor by a novel phosphoswitch.

Deleted in liver cancer 3 (DLC3) is a Rho GTPase-activating protein (RhoGAP) that plays a crucial role in maintaining adherens junction integrity and coordinating polarized vesicle transport by modulating Rho activity at the plasma membrane and endomembranes. By employing bioinformatical sequence analysis, in vitro experiments, and in cellulo assays we here identified a polybasic region (PBR) in DLC3 that facilitates the association of the protein with cellular membranes. Within the PBR, we mapped two serines whose phosphorylation can alter the electrostatic character of the region. Consequently, phosphomimetic mutations of these sites impaired the membrane association of DLC3. Furthermore, we found a new PBR-dependent localization of DLC3 at the midbody region, where the protein locally controlled Rho activity. Here, the phosphorylation-dependent regulation of DLC3 appeared to be required for proper cytokinesis. Our work thus provides a novel mechanism for spatiotemporal termination of Rho signaling by the RhoGAP protein DLC3.

A hybrid evaluation of the intestinal absorption performance of compounds from molecular structure.

Intestinal absorption of compounds is significant in drug research and development. To evaluate this efficiently, a method combining mathematical modeling and molecular simulation was proposed, from the perspective of molecular structure. Based on the quantitative structure-property relationship study, the model between molecular structure and their apparent permeability coefficients was successfully constructed and verified, predicting intestinal absorption of drugs and interpreting decisive structural factors, such as AlogP98, Hydrogen bond donor and Ellipsoidal volume. The molecules with strong lipophilicity, less hydrogen bond donors and receptors, and small molecular volume are more easily absorbed. Then, the molecular dynamics simulation and molecular docking were utilized to study the mechanism of differences in intestinal absorption of drugs and investigate the role of molecular structure. Results indicated that molecules with strong lipophilicity and small volume interacted with the membrane at a lower energy and were easier to penetrate the membrane. Likewise, they had weaker interaction with P-glycoprotein and were easier to escape from it and harder to export from the body. More in, less out, is the main reason these molecules absorb well.

Infection of Phytophthora palmivora Isolates on Arabidopsis thaliana.

Phytophthora palmivora, a hemibiotrophic oomycete, causes diseases in several economically important tropical crops, such as oil palm, which it is responsible for a devastating disease called bud rot (BR). Despite recent progress in understanding host resistance and virulence mechanisms, many aspects remain unknown in P. palmivora isolates from oil palm. Model pathosystems are useful for understanding the molecular interactions between pathogens and hosts. In this study, we utilized detached leaves and whole seedlings of Arabidopsis thaliana Col-0 to describe and evaluate the infection process of three P. palmivora isolates (CPPhZC-05, CPPhZC-04, CPPhZOC-01) that cause BR in oil palm. Two compatible isolates (CPPhZC-05 and CPPhZOC-01) induced aqueous lesions at 72 h post-inoculation (hpi), with microscopic visualization revealing zoospore encysting and appressorium penetration at 3 hpi, followed by sporangia generation at 72 hpi. In contrast, an incompatible isolate (CPPhZC-04) exhibited cysts that could not penetrate tissue, resulting in low leaf colonization. Gene expression of ten P. palmivora infection-related genes was quantified by RT-qPCR, revealing overexpression in compatible isolates, but not in the incompatible isolate. Additionally, key genes associated with salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) in Arabidopsis exhibited regulation during interaction with the three isolates. These findings demonstrate that P. palmivora can infect Arabidopsis Col-0, and variability is observed in the interaction between Arabidopsis-Col-0 and P. palmivora isolates. Establishing this pathosystem is expected to enhance our understanding of P. palmivora's pathology and physiology.

Interaction of plants and metal nanoparticles: Exploring its molecular mechanisms for sustainable agriculture and crop improvement.

Metal nanoparticles offer promising prospects in agriculture, enhancing plant growth and ensuring food security. Silver, gold, copper, and zinc nanoparticles possess unique properties making them attractive for plant applications. Understanding molecular interactions between metal nanoparticles and plants is crucial for unlocking their potential to boost crop productivity and sustainability. This review explores metal nanoparticles in agriculture, emphasizing the need to understand these interactions. By elucidating mechanisms, it highlights the potential for enhancing crop productivity, stress tolerance, and nutrient-use efficiency, contributing to sustainable agriculture and food security. Quantifying benefits and risks reveal significant advantages. Metal nanoparticles enhance crop productivity by 20% on average and reduce disease incidence by up to 50% when used as antimicrobial agents. They also reduce nutrient leaching by 30% and enhance soil carbon sequestration by 15%, but concerns about toxicity, adverse effects on non-target organisms, and nanoparticle accumulation in the food chain must be addressed. Metal nanoparticles influence cellular processes including sensing, signaling, transcription, translation, and post-translational modifications. They act as signaling molecules, activate stress-responsive genes, enhance defense mechanisms, and improve nutrient uptake. The review explores their catalytic role in nutrient management, disease control, precision agriculture, nano-fertilizers, and nano-remediation. A bibliometric analysis offers insights into the current research landscape, highlighting trends, gaps, and future directions. In conclusion, metal nanoparticles hold potential for revolutionizing agriculture, enhancing productivity, mitigating environmental stressors, and promoting sustainability. Addressing risks and gaps is crucial for their safe integration into agricultural practices.

Formation of pea protein amyloid-like nanofibrils-derived hydrogels mediated by epigallocatechin gallate.

This study investigated the interaction between pea protein amyloid-like nanofibril and epigallocatechin gallate, constructed and characterized the novel pea protein nanofibrils-derived hydrogel mediated by epigallocatechin gallate, and researched the functionalities of the hydrogel. Epigallocatechin gallate remodeled the structure of pea protein nanofibrils, and a stable and strong hydrogel was formed at a relatively low protein concentration (4.5%). Additionally, the hydrogels exhibited various surface structures and hydrogel properties dependent on the mass ratio. Strongest gel strength (51 g) was attained at 0.25 epigallocatechin gallate/pea protein nanofibrils mass ratio. Whereas, the hydrogels exhibited the highest water holding capacity (87%) at 0.05 mass ratio. The primary driving forces in the formation and maintaining of the hydrogels were hydrophobic interactions and ionic bonds. Progressive rise of ß-sheet content of pea protein nanofibrils occurred increasing epigallocatechin gallate concentration. This hydrogel holds great potential for applications in food processing, targeted delivery of nutraceuticals and biomedicine.