Microscale Thermophoresis Research Articles

Microscale Thermophoresis (MST) as a Tool to Study Binding Interactions of Oxygen-Sensitive Biohybrids.

Microscale thermophoresis (MST) is a technique used to measure the strength of molecular interactions. MST is a thermophoretic-based technique that monitors the change in fluorescence associated with the movement of fluorescent-labeled molecules in response to a temperature gradient triggered by an IR LASER. MST has advantages over other approaches for examining molecular interactions, such as isothermal titration calorimetry, nuclear magnetic resonance, biolayer interferometry, and surface plasmon resonance, requiring a small sample size that does not need to be immobilized and a high-sensitivity fluorescence detection. In addition, since the approach involves the loading of samples into capillaries that can be easily sealed, it can be adapted to analyze oxygen-sensitive samples. In this Bio-protocol, we describe the troubleshooting and optimization we have done to enable the use of MST to examine protein-protein interactions, protein-ligand interactions, and protein-nanocrystal interactions. The salient elements in the developed procedures include 1) loading and sealing capabilities in an anaerobic chamber for analysis using a NanoTemper MST located on the benchtop in air, 2) identification of the optimal reducing agents compatible with data acquisition with effective protection against trace oxygen, and 3) the optimization of data acquisition and analysis procedures. The procedures lay the groundwork to define the determinants of molecular interactions in these technically demanding systems. Key features • Established procedures for loading and sealing tubes in an anaerobic chamber for subsequent analysis. • Sodium dithionite (NaDT) could easily be substituted with one electron-reduced 1,1'-bis(3-sulfonatopropyl)-4,4'-bipyridinium [(SPr)2V•] to perform sensitive biophysical assays on oxygen-sensitive proteins like the MoFe protein. • Established MST as an experimental tool to quantify binding affinities in novel enzyme-quantum dot biohybrid complexes that are extremely oxygen-sensitive.

Validation of shikimate dehydrogenase as the herbicidal target of drupacine and screening of target-based compounds with high herbicidal activity

The discovery of new targets and lead compounds is the key to developing new pesticides. The herbicidal target of drupacine has been identified as shikimate dehydrogenase (SkDH). However, the mechanism of interaction between them remains unclear. This study found that drupacine specifically binds to SkDH with a dissociation equilibrium constant (KD) of 8.88 μM and a Kd value of 2.15 μM, as confirmed by surface plasmon resonance and microscale thermophoresis. Site-directed mutagenesis coupled with fluorescence quenching analysis indicated that residue THR431 was the key amino acid site for drupacine binding to SkDH. Nine compounds with the best binding ability to SkDH were identified by virtual screening from about 120,000 compounds. Among them, compound 8 showed the highest inhibition rate with values of 41.95% against SkDH, also exhibiting the strongest herbicidal activity. This research identifies a novel potential target SkDH and a candidate lead compound with high herbicidal activity for developing new herbicides.

Fucoxanthin Mitigates High-Fat-Induced Lipid Deposition and Insulin Resistance in Skeletal Muscle through Inhibiting PKM1 Activity.

Glucose and lipid metabolism dysregulation in skeletal muscle contributes to the development of metabolic disorders. The efficacy of fucoxanthin in alleviating lipid metabolic disorders in skeletal muscle remains poorly understood. In this study, we systematically investigated the impact of fucoxanthin on mitigating lipid deposition and insulin resistance in skeletal muscle employing palmitic acid-induced lipid deposition in C2C12 cells and ob/ob mice. Fucoxanthin significantly alleviated PA-induced skeletal muscle lipid deposition and insulin resistance. In addition, fucoxanthin prominently upregulated the expression of lipid metabolism-related genes (Pparα and Cpt-1), promoting fatty acid β-oxidation metabolism. Additionally, fucoxanthin significantly increased the expression of Pgc-1α and Tfam, elevated the mtDNA/nDNA ratio, and reduced ROS levels. Further, we identified pyruvate kinase muscle isozyme 1 (PKM1) as a high-affinity protein for fucoxanthin by drug affinity-responsive target stability and LC-MS and confirmed their robust interaction by CETSA, microscale thermophoresis, and circular dichroism. Supplementation with pyruvate, the product of PKM1, significantly attenuated the beneficial effects of fucoxanthin on lipid deposition and insulin resistance. Mechanistically, fucoxanthin reduced glucose glycolysis rate and enhanced mitochondrial biosynthesis and fatty acid β-oxidation through inhibiting PKM1 activity, thereby alleviating lipid metabolic stress. These findings present a novel clinical strategy for treating metabolic diseases using fucoxanthin.

Bioactive components in prunella vulgaris for treating Hashimoto's disease via regulation of innate immune response in human thyrocytes

BackgroundHashimoto's thyroiditis (HT) is a thyroid autoimmune disease characterized by lymphocytic infiltration and thyroid destruction. Prunella vulgaris (PV) is a traditional Chinese herbal medicine with documented clinical efficacy in treating HT. We previously reported an immunoregulatory effect of PV in thyrocytes; however, the bioactive components of PV remained unclear. This study aimed to elucidate key components of PV for treating HT and their acting mechanisms. MethodsNetwork pharmacology was used to predict key PV components for HT. The predicted components were tested to determine whether they could exert an immunoregulatory effect of PV in human thyrocytes. Limited proteolysis-mass spectrometry (Lip-MS) was used to explore interacting proteins with PV components in human thyrocytes. Microscale thermophoresis binding assay was used to evaluate the affinity of PV components with the target protein. ResultsEleven PV components with 192 component targets and 3415 HT-related genes were gathered from public databases. With network pharmacology, a ‘component-target-disease’ network was established wherein four flavonoids including quercetin, luteolin, kaempferol, morin, and a phytosterol, β-sitosterol were predicted as key components in PV for HT. In stimulated primary human thyrocytes or Nthy-ori-31 cells, key components inhibited gene expressions of inflammatory cytokines including tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and interferon-β (IFN-β), cellular apoptosis, and activation of nuclear factor κB (NF-κB) and interferon regulatory factor 3 (IRF-3). Heat shock protein 90 alpha, class A, member 1 (HSP90AA1), was identified to interact with flavonoids in PV by Lip-MS. Morin had the highest affinity with HSP90AA1 (KD = 122.74 μM), followed by kaempferol (KD = 168.53 μM), luteolin (KD = 293.94 μM), and quercetin (KD = 356.86 μM). ConclusionQuercetin, luteolin, kaempferol, morin, and β-sitosterol reproduced an anti-inflammatory and anti-apoptosis effect of PV in stimulated human thyrocytes, which potentially contributed to the treatment efficacy of PV in HT.

Formononetin attenuates hepatic injury in diabetic mice by regulating macrophage polarization through the PTP1B/STAT6 axis

BackgroundFormononetin (FNT) is an isoflavone known for its anti-inflammatory properties and has been shown to reduce insulin resistance in Type 2 Diabetes Mellitus (T2DM). However, its effects and the underlying mechanisms in diabetic liver injury remain largely unexplored. MethodsWe established a T2DM-induced liver injury mouse model by feeding high-fat diet, followed by injecting streptozotocin. The mice were then treated with FNT and the liver function in these mice was assessed. Macrophage markers in FNT-treated T2DM mice or human THP-1 cells were evaluated using flow cytometry, RT-qPCR, and Western blotting. The expression of PTP1B and STAT6 in mouse liver tissues and THP-1 cells was analyzed. Molecular docking predicted the interaction between PTP1B and STAT6, which was validated via co-immunoprecipitation (Co-IP) and phos-tag analysis. Microscale thermophoresis (MST) assessed the binding affinity of FNT to PTP1B. ResultsFNT treatment significantly ameliorated blood glucose levels, hepatocyte apoptosis, inflammatory response, and liver dysfunction in T2DM mice. Moreover, FNT facilitated M2 macrophage polarization in both T2DM mice and high glucose (HG)-induced THP-1-derived macrophages. The PTP1B/STAT6 axis, deregulated in T2DM mice, was normalized by FNT treatment, which counteracted the T2DM-induced upregulation of PTP1B and downregulation of phosphorylated STAT6. Molecular docking and subsequent analyses revealed that PTP1B binds to and dephosphorylates STAT6 at the S325A site. In contrast, FNT strongly binds to PTP1B and influences its expression at the K116A site, promoting M2 polarization of THP-1 cells via downregulation of PTP1B. ConclusionFormononetin mitigates diabetic hepatic injury by fostering M2 macrophage polarization via the PTP1B/STAT6 axis.

MnoSR removal in Mycobacterium smegmatis triggers broad transcriptional response to 1,3-propanediol and glucose as sole carbon sources.

The two-component signal transduction systems play an essential role in the adaptation of bacteria to changing environmental conditions. One of them is the MnoSR system involved in the regulation of methylotrophic metabolism in M. smegmatis. Mycobacterium smegmatis mutant strains ΔmnoS, ΔmnoR and ΔmnoS/R lacking functional mnoS, mnoR and both genes were generated using a homologous recombination approach. MnoR recombinant protein was purified by affinity column chromatography. The present study employs molecular biology techniques: cloning strategies, global RNA sequencing, qRT-PCR, EMSA, Microscale thermophoresis, and bioinformatics analysis. The ∆mnoS, ∆mnoR, and ∆mnoS/R mutant strains were generated and cultured in the presence of defined carbon sources. Growth curve analysis confirmed that inactivation of the MnoSR impairs the ability of M. smegmatis cells to use alcohols such as 1,3-propanediol and ethanol but improves the bacterial growth on ethylene glycol, xylitol, and glycerol. The total RNA sequencing method was employed to understand the importance of MnoSR in the global responses of mycobacteria to limited carbon access and in carbon-rich conditions. The loss of MnoSR significantly affected carbon utilization in the case of mycobacteria cultured on glucose or 1,3-propanediol as sole carbon sources as it influenced the expression of multiple metabolic pathways. The numerous transcriptional changes could not be linked to the presence of evident MnoR DNA-binding sites within the promotor regions for the genes outside of the mno operon. This was confirmed by EMSA and microscale thermophoresis with mutated MnoR binding consensus region. Our comprehensive analysis highlights the system's vital role in metabolic adaptability, providing insights into its potential impact on the environmental survival of mycobacteria.

Design, synthesis and antiviral activity of indole derivatives containing quinoline moiety.

A series of indole derivatives containing quinoline structures were designed and synthesized. The synthesized compounds were characterized by NMR and HRMS. And W14 was performed by single crystal X-ray diffraction experiments. The antiviral activity studies showed that some of the target compounds possessed significant activity against tobacco mosaic virus (TMV). In particular, W20 had significant activity. The results of in vivo anti-TMV activity assay showed that W20 possessed the best curative and protective activities with EC50 values of 84.4 and 65.7 μg/mL, which were better than ningnanmycin (NNM) 205.1 and 162.0 μg/mL, respectively. The results of Microscale thermophoresis (MST) showed that W20 had a strong binding affinity for the tobacco mosaic virus coat protein (TMV-CP) with a dissociation constant (Kd) of 0.00519 μmol/L, which was superior to that of NNM (1. 65320 μmol/L). The molecular docking studies were in accordance with the experimental results. In addition, the determination of malondialdehyde (MDA) content in tobacco leaves showed that W20 improved the disease resistance of tobacco. Overall, this study shows that indole derivatives containing quinoline can be used as new antiviral agents for plant viruses for further research.

Cinnamaldehyde activates AMPK/PGC-1α pathway via targeting GRK2 to ameliorate heart failure

BackgroundAccording to recent research, treating heart failure (HF) by inhibiting G protein-coupled receptor kinase 2 (GRK2) to improve myocardial energy metabolism has been identified as a potential approach. Cinnamaldehyde (CIN), a phenylpropyl aldehyde compound, has been demonstrated to exhibit beneficial effects in cardiovascular diseases. However, whether CIN inhibits GRK2 to ameliorate myocardial energy metabolism in HF is still unclear. PurposeThis study examines the effects of CIN on GRK2 and myocardial energy metabolism to elucidate its underlying mechanism to treat HF. MethodsThe isoproterenol (ISO) induced HF model in vivo and in vitro were constructed using Sprague-Dawley (SD) rats and primary neonatal rat cardiomyocytes (NRCMs). Based on this, the effects of CIN on myocardial energy metabolism and GRK2 were investigated. Additionally, validation experiments were conducted after interfering and over-expressing GRK2 in ISO-induced NRCMs to verify the regulatory effect of CIN on GRK2. Furthermore, binding capacity between GRK2 and CIN was explored by Cellular Thermal Shift Assay (CETSA) and Microscale Thermophoresis (MST). ResultsIn vivo and in vitro, CIN significantly improved HF as demonstrated by reversing abnormal changes in myocardial injury markers, inhibiting myocardial hypertrophy and decreasing myocardial fibrosis. Additionally, CIN promoted myocardial fatty acid metabolism to ameliorate myocardial energy metabolism disorder by activating AMPK/PGC-1α signaling pathway. Moreover, CIN reversed the inhibition of myocardial fatty acid metabolism and AMPK/PGC-1α signaling pathway by GRK2 over-expression in ISO-induced NRCMs. Meanwhile, CIN had no better impact on the stimulation of cardiac fatty acid metabolism and the AMPK/PGC-1α signaling pathway in ISO-induced NRCMs when GRK2 was disrupted. Noticeably, CETSA and MST confirmed that CIN binds to GRK2 directly. The binding of CIN and GRK2 promoted the ubiquitination degradation of GRK2 mediated by murine double mimute 2. ConclusionThis study demonstrates that CIN exerts a protective intervention in HF by targeting GRK2 and promoting its ubiquitination degradation to activate AMPK/PGC-1α signaling pathway, ultimately improving myocardial fatty acid metabolism.

Protein semisynthesis reveals plasticity in HECT E3 ubiquitin ligase mechanisms.

Lys ubiquitination is catalysed by E3 ubiquitin ligases and is central to the regulation of protein stability and cell signalling in normal and disease states. There are gaps in our understanding of E3 mechanisms, and here we use protein semisynthesis, chemical rescue, microscale thermophoresis and other biochemical approaches to dissect the role of catalytic base/acid function and conformational interconversion in HECT-domain E3 catalysis. We demonstrate that there is plasticity in the use of the terminal side chain or backbone carboxylate for proton transfer in HECT E3 ubiquitin ligase reactions, with yeast Rsp5 orthologues appearing to be possible evolutionary intermediates. We also show that the HECT-domain ubiquitin covalent intermediate appears to eject the E2 conjugating enzyme, promoting catalytic turnover. These findings provide key mechanistic insights into how protein ubiquitination occurs and provide a framework for understanding E3 functions and regulation.

Aniquinazoline B, a Fungal Natural Product, Activates the μ-Opioid Receptor.

The development of new μ-opioid receptor (MOR) agonists without the undesirable side effects, such as addiction or respiratory depression, has been a difficult challenge over the years. In the search for new compounds, we screened our chemical database of over 40.000 substances and further assessed the best 100 through molecular docking. We selected the top 10 compounds and evaluated them for their biological activity and potential to influence cyclic adenosine monophosphate (cAMP) levels. From the tested compounds, compound 7, called aniquinazoline B, belonging to the quinazolinone alkaloids class and isolated from the marine fungus Aspergillus nidulans, showed promising results, by inhibiting cAMP levels and in vitro binding to MOR, verified through microscale thermophoresis. Transcriptomic data investigation profiled the genes affected by compound 7 and discovered activation of different pathways compared to opioids. The western blot analysis revealed compound 7 as a balanced ligand, activating both p-ERK1/2 and β-arrestin1/2 pathways, showing this is a favorable candidate to be further tested.

Design, synthesis, and evaluation of antitumor activity of 2-arylmethoxy-4-(2-fluoromethyl-biphenyl-3-ylmethoxy) benzylamine derivatives as PD-1/PD-l1 inhibitors

A series of novel 2-arylmethoxy-4-(2-fluoromethyl-biphenyl-3-ylmethoxy) benzylamine derivatives was designed, synthesized, and evaluated for their antitumor effects as PD-1/PD-L1 inhibitors both in vitro and in vivo. Firstly, the ability of these compounds to block the PD-1/PD-L1 immune checkpoint was assessed using the homogeneous time-resolved fluorescence (HTRF) assay. Two of the compounds can strongly block the PD-1/PD-L1 interaction, with IC50 values of less than 10 nM, notably, compound HD10 exhibited significant clinical potential by inhibiting the PD-1/PD-L1 interaction with an IC50 value of 3.1 nM. Further microscale thermophoresis (MST) analysis demonstrated that HD10 had strong interaction with PD-L1 protein. Co-crystal structure (2.7 Å) analysis of HD10 in complex with the PD-L1 protein revealed a strong affinity between the compound and the target PD-L1 dimer. This provides a solid theoretical basis for further in vitro and in vivo studies. Next, a typical cell-based experiment demonstrated that HD10 could remarkably prevent the interaction of hPD-1 293 T cells from human recombinant PD-L1 protein, effectively restoring T cell function, and promoting IFN-γ secretion in a dose-dependent manner. Moreover, HD10 was effective in suppressing tumor growth (TGI = 57.31 %) in a PD-1/PD-L1 humanized mouse model without obvious toxicity. Flow cytometry, qPCR, and immunohistochemistry data suggested that HD10 inhibits tumor growth by activating the immune system in vivo. Based on these results, it seems likely that HD10 is a promising clinical candidate that should be further investigated.

Structural Basis for the Interaction between the Ezrin FERM-Domain and Human Aquaporins.

The Ezrin/Radixin/Moesin (ERM) family of proteins act as cross-linkers between the plasma membrane and the actin cytoskeleton. This mechanism plays an essential role in processes related to membrane remodeling and organization, such as cell polarization, morphogenesis and adhesion, as well as in membrane protein trafficking and signaling pathways. For several human aquaporin (AQP) isoforms, an interaction between the ezrin band Four-point-one, Ezrin, Radixin, Moesin (FERM)-domain and the AQP C-terminus has been demonstrated, and this is believed to be important for AQP localization in the plasma membrane. Here, we investigate the structural basis for the interaction between ezrin and two human AQPs: AQP2 and AQP5. Using microscale thermophoresis, we show that full-length AQP2 and AQP5 as well as peptides corresponding to their C-termini interact with the ezrin FERM-domain with affinities in the low micromolar range. Modelling of the AQP2 and AQP5 FERM complexes using ColabFold reveals a common mode of binding in which the proximal and distal parts of the AQP C-termini bind simultaneously to distinct binding sites of FERM. While the interaction at each site closely resembles other FERM-complexes, the concurrent interaction with both sites has only been observed in the complex between moesin and its C-terminus which causes auto-inhibition. The proposed interaction between AQP2/AQP5 and FERM thus represents a novel binding mode for extrinsic ERM-interacting partners.

The glycine-rich domain of GRP7 plays a crucial role in binding long RNAs and facilitating phase separation

Microscale thermophoresis (MST) is a well-established method to quantify protein-RNA interactions. In this study, we employed MST to analyze the RNA binding properties of glycine-rich RNA binding protein 7 (GRP7), which is known to have multiple biological functions related to its ability to bind different types of RNA. However, the exact mechanism of GRP7’s RNA binding is not fully understood. While the RNA-recognition motif of GRP7 is known to be involved in RNA binding, the glycine-rich region (known as arginine-glycine-glycine-domain or RGG-domain) also influences this interaction. To investigate to which extend the RGG-domain of GRP7 is involved in RNA binding, mutation studies on putative RNA interacting or modulating sites were performed. In addition to MST experiments, we examined liquid–liquid phase separation of GRP7 and its mutants, both with and without RNA. Furthermore, we systemically investigated factors that might affect RNA binding selectivity of GRP7 by testing RNAs of different sizes, structures, and modifications. Consequently, our study revealed that GRP7 exhibits a high affinity for a variety of RNAs, indicating a lack of pronounced selectivity. Moreover, we established that the RGG-domain plays a crucial role in binding longer RNAs and promoting phase separation.

Anti-Inflammatory and Cancer-Preventive Potential of Chamomile (Matricaria chamomilla L.): A Comprehensive In Silico and In Vitro Study.

Chamomile tea, renowned for its exquisite taste, has been appreciated for centuries not only for its flavor but also for its myriad health benefits. In this study, we investigated the preventive potential of chamomile (Matricaria chamomilla L.) towards cancer by focusing on its anti-inflammatory activity. A virtual drug screening of 212 phytochemicals from chamomile revealed β-amyrin, β-eudesmol, β-sitosterol, apigenin, daucosterol, and myricetin as potent NF-κB inhibitors. The in silico results were verified through microscale thermophoresis, reporter cell line experiments, and flow cytometric determination of reactive oxygen species and mitochondrial membrane potential. An oncobiogram generated through comparison of 91 anticancer agents with known modes of action using the NCI tumor cell line panel revealed significant relationships of cytotoxic chamomile compounds, lupeol, and quercetin to microtubule inhibitors. This hypothesis was verified by confocal microscopy using α-tubulin-GFP-transfected U2OS cells and molecular docking of lupeol and quercetin to tubulins. Both compounds induced G2/M cell cycle arrest and necrosis rather than apoptosis. Interestingly, lupeol and quercetin were not involved in major mechanisms of resistance to established anticancer drugs (ABC transporters, TP53, or EGFR). Performing hierarchical cluster analyses of proteomic expression data of the NCI cell line panel identified two sets of 40 proteins determining sensitivity and resistance to lupeol and quercetin, further pointing to the multi-specific nature of chamomile compounds. Furthermore, lupeol, quercetin, and β-amyrin inhibited the mRNA expression of the proinflammatory cytokines IL-1β and IL6 in NF-κB reporter cells (HEK-Blue Null1). Moreover, Kaplan-Meier-based survival analyses with NF-κB as the target protein of these compounds were performed by mining the TCGA-based KM-Plotter repository with 7489 cancer patients. Renal clear cell carcinomas (grade 3, low mutational rate, low neoantigen load) were significantly associated with shorter survival of patients, indicating that these subgroups of tumors might benefit from NF-κB inhibition by chamomile compounds. This study revealed the potential of chamomile, positioning it as a promising preventive agent against inflammation and cancer. Further research and clinical studies are recommended.

The Discovery of Highly Efficient and Promising ABA Receptor Antagonists for Agricultural Applications Based on APAn Modification.

Abscisic acid (ABA) is one of the many naturally occurring phytohormones widely found in plants. This study focused on refining APAn, a series of previously developed agonism/antagonism switching probes. Twelve novel APAn analogues were synthesized by introducing varied branched or oxygen-containing chains at the C-6' position, and these were screened. Through germination assays conducted on A. thaliana, colza, and rice seeds, as well as investigations into stomatal movement, several highly active ABA receptor antagonists were identified. Microscale thermophoresis (MST) assays, molecular docking, and molecular dynamics simulation showed that they had stronger receptor affinity than ABA, while PP2C phosphatase assays indicated that the C-6'-tail chain extending from the 3' channel effectively prevented the ligand-receptor binary complex from binding to PP2C phosphatase, demonstrating strong antagonistic activity. These antagonists showed effective potential in promoting seed germination and stomatal opening of plants exposed to abiotic stress, particularly cold and salt stress, offering advantages for cultivating crops under adverse conditions. Moreover, their combined application with fluridone and gibberellic acid could provide more practical agricultural solutions, presenting new insights and tools for overcoming agricultural challenges.

Arabidopsis PROTODERMAL FACTOR2 binds lysophosphatidylcholines and transcriptionally regulates phospholipid metabolism.

Plant homeodomain leucine zipper IV (HD-Zip IV) transcription factors (TFs) contain an evolutionarily conserved steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain. While the START domain is required for TF activity, its presumed role as a lipid sensor is not clear. Here we used tandem affinity purification from Arabidopsis cell cultures to demonstrate that PROTODERMAL FACTOR2 (PDF2), a representative member that controls epidermal differentiation, recruits lysophosphatidylcholines (LysoPCs) in a START-dependent manner. Microscale thermophoresis assays confirmed that a missense mutation in a predicted ligand contact site reduces lysophospholipid binding. We additionally found that PDF2 acts as a transcriptional regulator of phospholipid- and phosphate (Pi) starvation-related genes and binds to a palindromic octamer with consensus to a Pi response element. Phospholipid homeostasis and elongation growth were altered in pdf2 mutants according to Pi availability. Cycloheximide chase experiments revealed a role for START in maintaining protein levels, and Pi starvation resulted in enhanced protein destabilization, suggesting a mechanism by which lipid binding controls TF activity. We propose that the START domain serves as a molecular sensor for membrane phospholipid status in the epidermis. Our data provide insights toward understanding how the lipid metabolome integrates Pi availability with gene expression.

Netrin-1 Is an Important Mediator in Microglia Migration.

Microglia migrate to the cerebral cortex during early embryonic stages. However, the precise mechanisms underlying microglia migration remain incompletely understood. As an extracellular matrix protein, Netrin-1 is involved in modulating the motility of diverse cells. In this paper, we found that Netrin-1 promoted microglial BV2 cell migration in vitro. Mechanism studies indicated that the activation of GSK3β activity contributed to Netrin-1-mediated microglia migration. Furthermore, Integrin α6/β1 might be the relevant receptor. Single-cell data analysis revealed the higher expression of Integrin α6 subunit and β1 subunit in microglia in comparison with classical receptors, including Dcc, Neo1, Unc5a, Unc5b, Unc5c, Unc5d, and Dscam. Microscale thermophoresis (MST) measurement confirmed the high binding affinity between Integrin α6/β1 and Netrin-1. Importantly, activation of Integrin α6/β1 with IKVAV peptides mirrored the microglia migration and GSK3 activation induced by Netrin-1. Finally, conditional knockout (CKO) of Netrin-1 in radial glial cells and their progeny led to a reduction in microglia population in the cerebral cortex at early developmental stages. Together, our findings highlight the role of Netrin-1 in microglia migration and underscore its therapeutic potential in microglia-related brain diseases.

Exploring a new format of thermophoretic measurement – Stop-flow microscale thermophoresis in the narrow-bore transparent capillary

The classic format of the microscale thermophoresis technique (MST) requires the use of disposable glass capillaries. Their total capacity significantly exceeds the volume of the sample used directly in the thermophoretic measurement. An alternative approach that allows for reducing the consumption of sample, reagents and elimination of waste is the use of narrow-bore capillaries, commonly applied in the capillary electrophoresis technique (CE), in combination with injecting sample segment of a reduced length. This article presents the first-ever use of a transparent and flexible narrow-bore capillary for this purpose, which is much easier to handle than a regular non-transparent silica capillary. In this approach, it is possible to perform thermophoretic analysis with a small sample volume, which is delivered to the detection site using pressure-induced flow, or electrophoretically – while simultaneously performing electrophoretic separation. We focused primarily on the former approach, called Transparent Capillary Stop-Flow MicroScale Thermophoresis (TCSF-MST). Its potential was demonstrated in systems presenting the original use of the thermophoresis phenomenon: monitoring the progress of the fluorescent derivatization reaction, determining the critical micellization concentration of a surfactant, and quantitative analysis of fluorophores based on the internal standard method. The obtained results suggest that the new format can be considered an interesting alternative to the traditional MST methodology, and also seems consistent with the idea of “​​green” and “white” analytical chemistry.

Integrated bioinformatics and multiomics reveal Liupao tea extract alleviating NAFLD via regulating hepatic lipid metabolism and gut microbiota

BackgroundNon-alcoholic fatty liver disease (NAFLD) poses a significant global public health concern. Liupao tea (LPT) is a Chinese national geographical indication product renowned for its lipid-lowering properties. However, the precise mechanisms and active constituents contributing to the efficacy of LPT against NAFLD remain unclear. PurposeThis study aims to comprehensively explore the therapeutic potential of Liupao tea extract (LPTE) in alleviating NAFLD through an integrated strategy. MethodsInitially, network pharmacology analysis was conducted based on LPTE chemical ingredient analysis, identifying core targets and key components. Potential active ingredients were validated through chemical standards based on LC-MS/MS. To confirm the pharmacological efficacy of LPTE in NAFLD, NAFLD mice models were employed. Alterations in hepatic lipid metabolism were comprehensively elucidated through integration of metabolomics, lipidomics, network pharmacology analysis, and real-time PCR analysis. To further explore the binding interactions between key components and core targets, molecular docking and microscale thermophoresis (MST) analysis were employed. Furthermore, to investigate LPTE administration effectiveness on gut microbiota in NAFLD mice, a comprehensive approach was employed. This included Metorigin analysis, 16S rRNA sequencing, molecular docking, and fecal microbiome transplantation (FMT). ResultsStudy identified naringenin, quercetin, luteolin, and kaempferol as the potential active ingredients of LPTE. These compounds exhibited therapeutic potential for NAFLD by targeting key proteins such as PTGS2, CYP3A4, and ACHE, which are involved in the metabolic pathways of hepatic linoleic acid (LA) and glycerophospholipid (GP) metabolism. The therapeutic effectiveness of LPTE was observed to be comparable to that of simvastatin. Furthermore, LPTE exhibited notable efficacy in alleviating NAFLD by influencing alterations in gut microbiota composition (Proteobacteria phylum, Lactobacillus and Dubosiella genus) that perhaps impact LA and GP metabolic pathways. ConclusionLPTE could be effective in preventing high-fat diet (HFD)-induced NAFLD by modulating hepatic lipid metabolism and gut microbiota. This study firstly integrated bioinformatics and multi-omics technologies to identify the potential active components and key microbiota associated with LPTE's effects, while also primally elucidating the action mechanisms of LPTE in alleviating NAFLD. The findings offer a conceptual basis for LPTE's potential transformation into an innovative pharmaceutical agent for NAFLD prevention.

Ferulic Acid Dimers as Potential Antiviral Agents by Inhibiting TMV Self-Assembly.

A series of ferulic acid dimers were designed, synthesized, and evaluated for anti-TMV activity. Biological assays demonstrated that compounds A6, E3, and E5 displayed excellent inactivating against tobacco mosaic virus (TMV) with EC50 values of 62.8, 94.4, and 85.2 μg mL-1, respectively, which were superior to that of ningnanmycin (108.1 μg mL-1). Microscale thermophoresis indicated that compounds A6, E3, and E5 showed strong binding capacity to TMV coat protein with binding affinity values of 1.862, 3.439, and 2.926 μM, respectively. Molecular docking and molecular dynamics simulation revealed that compound A6 could firmly bind to the TMV coat protein through hydrogen and hydrophobic bonds. Transmission electron microscopy and self-assembly experiments indicated that compound A6 obviously destroyed the integrity of the TMV particles and blocked the virus from infecting the host. This study revealed that A6 can be used as a promising leading structure for the development of antiviral agents by inhibiting TMV self-assembly.