Enhanced brain-targeting and efficacy of cannabidiol via RVG-Exo/CBD nanodelivery system.

This study introduces an innovative brain-targeted drug delivery system, RVG-Exo/CBD, utilizing rabies virus glycoprotein (RVG)-engineered exosomes for encapsulating cannabidiol (CBD). The novel delivery system was meticulously characterized, confirming the maintenance of exosomal integrity, size, and successful drug encapsulation with a high drug loading rate of 83.0 %. Evaluation of the RVG-Exo/CBD's brain-targeting capability demonstrated superior distribution and retention in brain tissue compared to unmodified exosomes, primarily validated through in vivo fluorescence imaging. The efficacy of this delivery system was assessed using a behavioral sensitization model in mice, where RVG-Exo/CBD notably suppressed methamphetamine-induced hyperactivity more effectively than CBD alone, indicating a reduction in effective dose and enhanced bioavailability. Overall, the RVG-Exo/CBD system emerges as a promising strategy for enhancing the therapeutic efficacy and safety of CBD, particularly for neurological applications, highlighting its potential for addressing the limitations associated with traditional CBD administration in clinical settings.

Cannabidivarin alleviates α-synuclein aggregation via DAF-16 in Caenorhabditis elegans.

Cannabidivarin (CBDV), a structural analog of cannabidiol (CBD), has received attention in recent years owing to its anticonvulsant property and potential for treating autism spectrum disorder. However, the function and mechanism of CBDV involved in the progression of Parkinson's disease (PD) remain unclear. In this work, we found that CBDV inhibited α-synuclein (α-syn) aggregation in an established transgenetic Caenorhabditis elegans (C. elegans). The phenolic hydroxyl groups of CBDV are critical for scavenging reactive oxygen species (ROS), reducing the in vivo aggregation of α-syn and preventing DAergic neurons from 6-hydroxydopamine (6-OHDA)-induced injury and degeneration. By combining multiple biophysical approaches, including nuclear magnetic resonance spectrometry, transmission electron microscopy and fibrillation kinetics assays, we confirmed that CBDV does not directly interact with α-syn or inhibit the formation of α-syn fibrils in vitro. Further cellular signaling investigation showed that the ability of CBDV to prevent oxidative stress, the accumulation of α-syn and the degeneration of DAergic neurons was mediated by DAF-16 in the worms. This study demonstrates that CBDV alleviates the aggregation of α-syn in vivo and reveals that the phenolic hydroxyl groups of CBDV are critical for this activity, providing a potential for the development of CBDV as a drug candidate for PD therapeutics.

Cannabidiol alleviates neuroinflammation and attenuates neuropathic pain via targeting FKBP5.

Microglia is a heterogeneous population that mediates neuroinflammation in the central nervous system (CNS) and plays a crucial role in developing neuropathic pain. FKBP5 facilitates the assembly of the IκB kinase (IKK) complex for the activation of NF-κB, which arises as a novel target for treating neuropathic pain. In this study, cannabidiol (CBD), a main active component of Cannabis, was identified as an antagonist of FKBP5. In vitro protein intrinsic fluorescence titration showed that CBD directly bound to FKBP5. Cellular thermal shift assay (CETSA) indicated that CBD binding increased the FKBP5 stability, which implies that FKBP5 is the endogenous target of CBD. CBD was found to inhibit the assembly of the IKK complex and the activation of NF-κB, therefore blocking LPS-induced NF-κB downstream pro-inflammatory factors NO, IL-1ß, IL-6 and TNF-α. Stern-Volmer analysis and protein thermal shift assay revealed that tyrosine 113 (Y113) of FKBP5 was critical for FKBP5 interacting with CBD, which is consistent with in silico molecular docking simulation. FKBP5 Y113 mutation (Y113A) alleviated the effect of CBD inhibiting LPS-induced pro-inflammatory factors overproduction. Furthermore, systemic administration of CBD inhibited chronic constriction injury (CCI)-induced microglia activation and FKBP5 overexpression in lumbar spinal cord dorsal horn. These data imply that FKBP5 is an endogenous target of CBD.

Exploring the trimerization process of a transmembrane helix with an ionizable residue by molecular dynamics simulations: a case study of transmembrane domain 5 of LMP-1.

The oligomerization of membrane proteins is an important biological process that plays a critical role in the initialization of membrane protein receptor signaling. Unveiling how transmembrane segments oligomerize is critical for understanding the mechanism of membrane receptor signaling activation. Owing to the complicated membrane environment and the extraordinary dynamic properties of the ionizable residues in the transmembrane segment, it is extremely challenging to thoroughly understand the oligomerization process of the transmembrane domain. In this study, transmembrane domain 5 (TMD5) of latent membrane protein-1 from Epstein-Barr virus was used as a prototype model to investigate the trimerization process of the transmembrane segment with ionizable residues. The trimerization process of TMD5 was rebuilt and investigated via conventional molecular dynamics simulations and constant-pH molecular dynamics simulations. When TMD5s approached each other, the tilting angles of the TMD5 monomer decreased. TMD5s formed stable trimers until two interacting sites (D150s and Q139s) along each transmembrane helix were created to lock the TMD5s. The pKa values of D150 shifted toward neutral states in the membrane environment. When TMD5s were monomers, the pKa shift of D150 was mainly influenced by its microenvironment in the lipid bilayer. When TMD5s were moving close to each other, protein-protein interactions became the main contributing factor for the pKa shift of D150s. Overall, this work elucidates the behavior of the TMD5 helix and the pKa shift of ionizable residue D150 in the process of TMD5 oligomerization. This study may provide insight into the development of agents for targeting the oligomerization of membrane proteins.

Artemisinin inhibits TLR4 signaling by targeting co-receptor MD2 in microglial BV-2 cells and prevents lipopolysaccharide-induced blood-brain barrier leakage in mice.

Artemisinin and its derivatives have been the frontline drugs for treating malaria. In addition to the antiparasitic effect, accumulating evidence shows that artemisinins can alleviate neuroinflammatory responses in the central nervous system (CNS). However, the precise mechanisms underlying their anti-neuroinflammatory effects are unclear. Herein we attempted to delineate the molecule target of artemisinin in microglia. In vitro protein intrinsic fluorescence titrations and saturation transfer difference (STD)-NMR showed the direct binding of artemisinin to Toll-like receptor TLR4 co-receptor MD2. Cellular thermal shift assay (CETSA) showed that artemisinin binding increased MD2 stability, which implies that artemisinin directly binds to MD2 in the cellular context. Artemisinin bound MD2 showed much less collapse during the molecular dynamic simulations, which supports the increased stability of MD2 upon artemisinin binding. Flow cytometry analysis showed artemisinin inhibited LPS-induced TLR4 dimerization and endocytosis in microglial BV-2 cells. Therefore, artemisinin was found to inhibit the TLR4-JNK signaling axis and block LPS-induced pro-inflammatory factors nitric oxide, IL-1ß and TNF-α in BV-2 cells. Furthermore, artemisinin restored LPS-induced decrease of junction proteins ZO-1, Occludin and Claudin-5 in primary brain microvessel endothelial cells, and attenuated LPS-induced blood-brain barrier disruption in mice as assessed by Evans blue. In all, this study unambiguously adds MD2 as a direct binding target of artemisinin in its anti-neuroinflammatory function. The results also suggest that artemisinin could be repurposed as a potential therapeutic intervention for inflammatory CNS diseases.

Stereochemistry and innate immune recognition: (+)-norbinaltorphimine targets myeloid differentiation protein 2 and inhibits toll-like receptor 4 signaling.

Deregulation of innate immune TLR4 signaling contributes to various diseases including neuropathic pain and drug addiction. Naltrexone is one of the rare TLR4 antagonists with good blood-brain barrier permeability and showing no stereoselectivity for TLR4. By linking 2 naltrexone units through a rigid pyrrole spacer, the bivalent ligand norbinaltorphimine was formed. Interestingly, (+)-norbinaltorphimine [(+)-1] showed ∼25 times better TLR4 antagonist activity than naltrexone in microglial BV-2 cell line, whereas (-)-norbinaltorphimine [(-)-1] lost TLR4 activity. The enantioselectivity of norbinaltorphimine was further confirmed in primary microglia, astrocytes, and macrophages. The activities of meso isomer of norbinaltorphimine and the molecular dynamic simulation results demonstrate that the stereochemistry of (+)-1 is derived from the (+)-naltrexone pharmacophore. Moreover, (+)-1 significantly increased and prolonged morphine analgesia in vivo. The efficacy of (+)-1 is long lasting. This is the first report showing enantioselective modulation of the innate immune TLR signaling.-Zhang, X., Peng, Y., Grace, P. M., Metcalf, M. D., Kwilasz, A. J., Wang, Y., Zhang, T., Wu, S., Selfridge, B. R., Portoghese, P. S., Rice, K. C., Watkins, L. R., Hutchinson, M. R., Wang, X. Stereochemistry and innate immune recognition: (+)-norbinaltorphimine targets myeloid differentiation protein 2 and inhibits toll-like receptor 4 signaling.

Exploring Methamphetamine Nonenantioselectively Targeting Toll-like Receptor 4/Myeloid Differentiation Protein 2 by in Silico Simulations and Wet-Lab Techniques.

Methamphetamine (METH) is one of the highly addictive nonopioid psychostimulants, acting as a xenobiotic-associated molecular pattern (XAMP) to target TLR4 and activate microglia. However, the molecule recognition of METH by innate immune receptor TLR4/MD-2 is not well-understood. METH exists in two enantiomeric forms, and it is unclear whether the TLR4 innate immune recognition with METH is stereoselective. Herein, molecular dynamics (MDs) simulations were performed to dissect the recognition of (+)-METH and (-)-METH by TLR4/MD-2 at the atomic level. Amphetamine (AMPH), which is an analogue of METH, was also investigated for comparison. Computational simulations indicate that METH binds into the interaction interface between MD-2 as well as TLR4* that is from the adjacent copy of TLR4-MD-2, therefore stabilizing the active heterotetramer (TLR4/MD-2)2 complex. The calculated binding free energies and potential of mean force (PMF) values show that (-)-METH and (+)-METH have similar TLR4/MD-2 binding affinity. Further dynamics analyses of bindings with TLR4/MD-2 indicate that (-)-METH and (+)-METH behave similarly. Unlike the stereoselective neuron-stimulating activities of METH, no enantioselectivity was observed for METH interacting with TLR4/MD-2 complex as well as activating TLR4 signaling. Compared to METH, AMPH showed much weaker interactions with TLR4/MD-2, indicating that the substituted methyl group is critical in the molecular recognition of METH by TLR4/MD-2. In all, this study provides molecular insight into the innate immune recognition of METH, which demonstrates that METH could be nonenantioselectively sensed by TLR4/MD-2.

Lovastatin inhibits Toll-like receptor 4 signaling in microglia by targeting its co-receptor myeloid differentiation protein 2 and attenuates neuropathic pain.

There is growing interest in drug repositioning to find new therapeutic indications for drugs already approved for use in people. Lovastatin is an FDA approved drug that has been used clinically for over a decade as a lipid-lowering medication. While lovastatin is classically considered to act as a hydroxymethylglutaryl (HMG)-CoA reductase inhibitor, the present series of studies reveal a novel lovastatin effect, that being as a Toll-like receptor 4 (TLR4) antagonist. Lovastatin selectively inhibits lipopolysaccharide (LPS)-induced TLR4-NF-κB activation without affecting signaling by other homologous TLRs. In vitro biophysical binding and cellular thermal shift assay (CETSA) show that lovastatin is recognized by TLR4's coreceptor myeloid differentiation protein 2 (MD-2). This finding is supported by molecular dynamics simulations that lovastatin targets the LPS binding pocket of MD-2 and lovastatin binding stabilizes the MD-2 conformation. In vitro studies of BV-2 microglial cells revealed that lovastatin inhibits multiple effects of LPS, including activation of NFkB; mRNA expression of tumor necrosis factor-a, interleukin-6 and cyclo-oxygenase 2; production of nitric oxide and reactive oxygen species; as well as phagocytic activity. Furthermore, intrathecal delivery of lovastatin over lumbosacral spinal cord of rats attenuated both neuropathic pain from sciatic nerve injury and expression of the microglial activation marker CD11 in lumbar spinal cord dorsal horn. Given the well-established role of microglia and proinflammatory signaling in neuropathic pain, these data are supportive that lovastatin, as a TLR4 antagonist, may be productively repurposed for treating chronic pain.

Sorafenib targets the mitochondrial electron transport chain complexes and ATP synthase to activate the PINK1-Parkin pathway and modulate cellular drug response.

Sorafenib (Nexavar) is a broad-spectrum multikinase inhibitor that proves effective in treating advanced renal-cell carcinoma and liver cancer. Despite its well-characterized mechanism of action on several established cancer-related protein kinases, sorafenib causes variable responses among human tumors, although the cause for this variation is unknown. In an unbiased screening of an oncology drug library, we found that sorafenib activates recruitment of the ubiquitin E3 ligase Parkin to damaged mitochondria. We show that sorafenib inhibits the activity of both complex II/III of the electron transport chain and ATP synthase. Dual inhibition of these complexes, but not inhibition of each individual complex, stabilizes the serine-threonine protein kinase PINK1 on the mitochondrial outer membrane and activates Parkin. Unlike the protonophore carbonyl cyanide m-chlorophenylhydrazone, which activates the mitophagy response, sorafenib treatment triggers PINK1/Parkin-dependent cellular apoptosis, which is attenuated upon Bcl-2 overexpression. In summary, our results reveal a new mechanism of action for sorafenib as a mitocan and suggest that high Parkin activity levels could make tumor cells more sensitive to sorafenib's actions, providing one possible explanation why Parkin may be a tumor suppressor gene. These insights could be useful in developing new rationally designed combination therapies with sorafenib.

Dissecting the Innate Immune Recognition of Opioid Inactive Isomer (+)-Naltrexone Derived Toll-like Receptor 4 (TLR4) Antagonists.

The opioid inactive isomer (+)-naltrexone is one of the rare Toll-like receptor 4 (TLR4) antagonists with good blood-brain barrier (BBB) permeability, which is a lead with promising potential for treating neuropathic pain and drug addiction. (+)-Naltrexone targets the lipopolysaccharides (LPS) binding pocket of myeloid differentiation protein 2 (MD-2) and blocks innate immune TLR4 signaling. However, the details of the molecular interactions of (+)-naltrexone and its derivatives with MD-2 are not fully understood, which hinders the ligand-based drug discovery. Herein, in silico and in vitro assays were performed to elucidate the innate immune recognition of the opioid inactive (+)-isomers. The results showed that the conserved LPS binding pocket of MD-2 accommodated these opioid inactive (+)-isomers. The calculated binding free energies of (+)-naltrexone and its derivatives in complex with MD-2 correlated well with their experimental binding affinities and TLR4 antagonistic activities. Hydrophobic residues in the MD-2 cavity interacted directly with these (+)-naltrexone based TLR4 antagonists and principally participated in ligand binding. Increasing the hydrophobicity of substituted group at N-17 improved its TLR4 antagonistic activity, while charged groups disfavored the binding with MD-2. Molecular dynamics (MD) simulations showed the binding of (+)-naltrexone or its derivatives to MD-2 stabilized the "collapsed" conformation of MD-2, consequently blocking the binding and signaling of TLR4. Thermodynamics and dynamic analysis showed the topology of substituted group at N-17 of (+)-naltrexone affected the binding with MD-2 and TLR4 antagonistic activity. This study provides a molecular insight into the innate immune recognition of opioid inactive (+)-isomers, which would be of great help for the development of next-generation of (+)-opioid based TLR4 antagonists.

Preparative Separation and Purification of Four Glycosides from Gentianae radix by High-Speed Counter-Current Chromatography and Comparison of Their Anti-NO Production Effects.

Secoiridoid and iridoid glycosides are the main active components of Gentianaeradix. In this work, one iridoid and three secoiridoid glycosides from Gentianaeradix have been purified by high-speed counter-current chromatography in two runs using different solvent systems. Ethyl acetate-n-butanol-water (2:1:3, v/v/v) was the optimum solvent system to purify ca. 4.36 mg of loganic acid, 3.05 mg of swertiamarin, and 35.66 mg of gentiopicroside with 98.1%, 97.2% and 98.6% purities, respectively, while 31.15 mg of trifloroside with 98.9% purity was separated using hexane-ethyl acetate-methanol-water (1:3:1:3, v/v/v/v). The structures of the glycosides were identified by mass spectrometry and NMR. After separation, the anti-nitric oxide production effects of the compounds on lipopolysaccharide-induced BV-2 murine microglial cells were also evaluated. All of the compounds inhibited the production of nitric oxide in lipopolysaccharide-induced BV-2 cells with high cell viabilities in a concentration-dependent manner, which demonstrated that were able to be used as a nitric oxide inhibitor.

Transcriptome analysis of sika deer in China.

Sika deer is of great commercial value because their antlers are used in tonics and alternative medicine and their meat is healthy and delicious. The goal of this study was to generate transcript sequences from sika deer for functional genomic analyses and to identify the transcripts that demonstrate tissue-specific, age-dependent differential expression patterns. These sequences could enhance our understanding of the molecular mechanisms underlying sika deer growth and development. In the present study, we performed de novo transcriptome assembly and profiling analysis across ten tissue types and four developmental stages (juvenile, adolescent, adult, and aged) of sika deer, using Illumina paired-end tag (PET) sequencing technology. A total of 1,752,253 contigs with an average length of 799 bp were generated, from which 1,348,618 unigenes with an average length of 590 bp were defined. Approximately 33.2 % of these (447,931 unigenes) were then annotated in public protein databases. Many sika deer tissue-specific, age-dependent unigenes were identified. The testes have the largest number of tissue-enriched unigenes, and some of them were prone to develop new functions for other tissues. Additionally, our transcriptome revealed that the juvenile-adolescent transition was the most complex and important stage of the sika deer life cycle. The present work represents the first multiple tissue transcriptome analysis of sika deer across four developmental stages. The generated data not only provide a functional genomics resource for future biological research on sika deer but also guide the selection and manipulation of genes controlling growth and development.

Protective Effects of Velvet Antler Methanol Extracts on Hypoxia-Induced Damage in Caenorhabditis elegans through HIF-1 and ECH-8 Mediated Lipid Accumulation.

Velvet antler, a traditional tonic widely used in East Asia for its health benefits, is explored in this study for its protective effects against hypoxia-induced damage using Caenorhabditis elegans (C. elegans) as a model. Hypoxia, characterized by low oxygen availability, induces significant physiological stress and potential tissue damage. Our research demonstrates that methanol extracts from velvet antler (MEs) enhance the survival of C. elegans under hypoxic conditions. This enhancement is achieved through the stabilization of hypoxia-inducible factor-1 (HIF-1) and the promotion of lipid accumulation, both of which are crucial for mitigating cellular damage. Specifically, MEs improve mitochondrial function, increase ATP production, and aid in the recovery of physical activity in C. elegans post-hypoxia or following hypoxia-reoxygenation (HR). The pivotal role of HIF-1 is underscored by the loss of these protective effects when HIF-1 function is inhibited. Additionally, our findings reveal that the gene related to lipid metabolism, ech-8, significantly contributes to the lipid accumulation that enhances resilience to hypoxia in C. elegans treated with MEs. These results not only highlight the therapeutic potential of velvet antler in modern medical applications, particularly for conditions involving hypoxic stress, but also provide insights into the molecular mechanisms by which MEs confer protection against hypoxic damage.

Quality and efficiency assessment of five extracellular vesicle isolation methods using the resistive pulse sensing strategy.

Extracellular vesicles (EVs) have attracted great interest due to their great potential in disease diagnosis and therapy. The separation of EVs from complex biofluids with high purity is essential for the accurate analysis of EVs. Despite various methods, there is still no consensus on the best method for high-quality EV isolation and reliable mass production. Therefore, it is important to offer a standardized method for characterizing the properties (size distribution, particle concentration and purity) of EV preparations from different isolation methods. Herein, we employed a NanoCoulter Counter based on the resistive pulse sensing (RPS) strategy that enabled multi-parameter analysis of single EVs to compare the quality and efficiency of different EV isolation techniques including traditional differential ultracentrifugation, ultrafiltration, size exclusion chromatography, membrane affinity binding and polymer precipitation. The data revealed that the NanoCoulter Counter based on the RPS strategy was reliable and effective for the characterization of EVs. The results suggested that although higher particle concentrations were observed in three commercial isolation kits and ultrafiltration, traditional differential ultracentrifugation showed the highest purity. In conclusion, our results from the NanoCoulter Counter provided reliable evidence for the assessment of different EV isolation methods, which contributed to the development of EV-based disease biomarkers and treatments.

Sika Deer Velvet Antler Peptide Exerts Neuroprotective Effect in a Parkinson's Disease Model via Regulating Oxidative Damage and Gut Microbiota.

Parkinson's disease (PD) is the second most common neurodegenerative disorder globally. Recognizing the potential of velvet antler in the nervous system, as shown in numerous studies, this research was aimed at evaluating the neuroprotective effects of Sika Deer velvet antler peptide (VAP), along with the underlying mechanisms in neurotoxin-induced PD models. Initially, a peptidomic analysis of the VAP, which comprised 189 varieties of peptides, was conducted using LC-MS. Nine sequences were identified as significant using Proteome Discoverer 2.5 software. In a cellular model of PD, where PC12 cells are treated with the neurotoxin 1-methyl-4-phenylpyridinium (MPP+), the administration of the VAP reduced the cell damage and apoptosis induced by MPP+. This protective effect was associated with a decrease in oxidative stress. This protective mechanism was found to be mediated through the activation of the SIRT1-dependent Akt/Nrf2/HO-1-signaling pathway. In animal models, specifically in mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD, the administration of the VAP effectively reduced the dopaminergic neuron damage and reversed the neurobehavioral deficits. They also diminished microglia activation and apoptosis, all without any noticeable adverse effects. Additionally, the VAP was observed to beneficially alter the gut microbiota, as marked by an increase in the abundances of Prevotellaceae, Helicobacteraceae, and Prevotella. These findings suggest that VAP exerts its neuroprotective effect against neurodegeneration by inhibiting oxidative stress and modulating gut microbiota.

GSK3 regulation Wnt/ß-catenin signaling affects adipogenesis in bovine skeletal muscle fibro/adipogenic progenitors.

Clarifying the cellular origin and regulatory mechanisms of intramuscular fat (IMF) deposition is crucial for improving beef quality. Here, we used single-nucleus RNA sequencing to analyze the structure and heterogeneity of skeletal muscle cell populations in different developmental stages of Yanbian cattle and identified eight cell types in two developmental stages of calves and adults. Among them, fibro/adipogenic progenitors (FAPs) expressing CD29 (ITGA7)pos and CD56 (NCAM1)neg surface markers were committed to IMF deposition in beef cattle and expressed major Wnt ligands and receptors. LY2090314/XAV-939 was used to activate/inhibit Wnt/ß-catenin signal. The results showed that the blockade of Glycogen Synthase Kinase 3 (GSK3) by LY2090314 promoted the stabilization of ß-catenin and reduced the expression of genes related adipogenic differentiation (e.g., PPARγ and C/EBPα) in bovine FAPs, confirming the anti-adipogenic effect of GSK3. XAV-939 inhibition of the Wnt/ß-catenin pathway promoted the lipid accumulation capacity of FAPs. Furthermore, we found that blocking GSK3 enhanced the paracrine effects of FAPs-MuSCs and increased myotube formation in muscle satellite cells (MuSCs). Overall, our results outline a single-cell atlas of skeletal muscle development in Yanbian cattle, revealed the role of Wnt/GSK3/ß-catenin signaling in FAPs adipogenesis, and provide a theoretical basis for further regulation of bovine IMF deposition.

GALNT12 promotes fibrosarcoma growth by accelerating YAP1 nuclear localization.

Fibrosarcoma is a highly malignant type of soft tissue sarcoma that currently lacks effective treatment options. Polypeptide N-acetylgalactosaminyltransferase 12 (GALNT12) belongs to the uridine diphosphate N-acetylgalactosamine gene family, which is involved in numerous biological processes of diseases, such as tumor progression. Its upregulated expression is closely associated with the development of colorectal cancer. However, research on the role of GALNT12 in fibrosarcoma is currently limited. The present study aimed to assess the expression and biological function of GALNT12 in fibrosarcoma. Patient data and tissue samples were collected and public datasets were obtained from the Gene Expression Omnibus (GSE24369 and GSE21124). Immunofluorescence assays were performed to observe the cellular localization of GALNT12. GALNT12 expression was measured using reverse transcription-quantitative PCR, western blotting and immunohistochemistry. Small interfering RNAs were constructed to knock down GALNT12 expression in HT-1080 cells. Cell Counting Kit-8 and EdU assays were used to assess fibrosarcoma cell proliferation. Wound healing and Transwell assays were used to detect migration. Gene set enrichment analysis was performed to identify key pathways. Paired and unpaired Student's t-test, Fisher's exact test and one-way ANOVA (followed by Tukey's Honest Significant Difference test) were used to analyze the data. It was demonstrated that GALNT12 expression was upregulated in both fibrosarcoma cell lines and tissue samples and predicted poor patient prognosis. In vitro experiments demonstrated that high GALNT12 expression levels significantly increased HT-1080 cell proliferation and migration. Furthermore, it was demonstrated that high GALNT12 expression levels were closely associated with the yes1 associated transcriptional regulator (YAP1) signaling pathway. Knockdown of GALNT12 inhibited YAP1 nuclear translocation, which affected activation of key downstream genes including AMOTL2, BIRC5 and CYR61. Therefore, the present study demonstrated that GALNT12 promoted fibrosarcoma progression. GALNT12 could be a potential biomarker for this disease and may potentially provide new ideas for targeted therapy of fibrosarcoma in the future.

Cannabidiol Analogue CIAC001 for the Treatment of Morphine-Induced Addiction by Targeting PKM2.

Opioid addiction is a chronically relapsing disorder that causes critical public health problems. Currently, there is a lack of effective drug treatment. Herein, one cannabidiol derivative, CIAC001, was discovered as an effective agent for treating morphine-induced addiction. In vitro, CIAC001 exhibited significantly improved anti-neuroinflammatory activity with lower toxicity. In vivo, CIAC001 ameliorated the morphine-induced withdrawal reaction, behavioral sensitization, and conditional position preference by inhibiting morphine-induced microglia activation and neuroinflammation. Target fishing for CIAC001 by activity-based protein profiling led to the identification of pyruvate kinase M2 (PKM2) as the target protein. CIAC001 bound to the protein-protein interface of the PKM2 dimer and promoted the tetramerization of PKM2. Moreover, CIAC001 exhibited an anti-neuroinflammatory effect by reversing the decrease of the PKM2 tetramer and inhibiting the nuclear translocation of PKM2. In summary, this study identified CIAC001 as a lead compound in targeting PKM2 to treat morphine-induced addiction.

ACT001 Inhibits TLR4 Signaling by Targeting Co-Receptor MD2 and Attenuates Neuropathic Pain.

Neuropathic pain is a common and challenging neurological disease, which renders an unmet need for safe and effective new therapies. Toll-like receptor 4 (TLR4) expressed on immune cells in the central nervous system arises as a novel target for treating neuropathic pain. In this study, ACT001, an orphan drug currently in clinical trials for the treatment of glioblastoma, was identified as a TLR4 antagonist. In vitro quenching titrations of intrinsic protein fluorescence and saturation transfer difference (STD)-NMR showed the direct binding of ACT001 to TLR4 co-receptor MD2. Cellular thermal shift assay (CETSA) showed that ACT001 binding affected the MD2 stability, which implies that MD2 is the endogenous target of ACT001. In silico simulations showed that ACT001 binding decreased the percentage of hydrophobic area in the buried solvent-accessible surface areas (SASA) of MD2 and rendered most regions of MD2 to be more flexible, which is consistent with experimental data that ACT001 binding decreased MD2 stability. In keeping with targeting MD2, ACT001 was found to restrain the formation of TLR4/MD2/MyD88 complex and the activation of TLR4 signaling axes of NF-κB and MAPKs, therefore blocking LPS-induced TLR4 signaling downstream pro-inflammatory factors NO, IL-6, TNF-α, and IL-1ß. Furthermore, systemic administration of ACT001 attenuated allodynia induced by peripheral nerve injury and activation of microglia and astrocyte in vivo. Given the well-established role of neuroinflammation in neuropathic pain, these data imply that ACT001 could be a potential drug candidate for the treatment of chronic neuropathic pain.

C. elegans as an in vivo model system for the phenotypic drug discovery for treating paraquat poisoning.

BACKGROUND: Paraquat (PQ) is an effective and widely used herbicide and causes numerous fatalities by accidental or voluntary ingestion. However, neither the final cytotoxic mechanism nor effective treatments for PQ poisoning have been discovered. Phenotypic drug discovery (PDD), which does not rely on the molecular mechanism of the diseases, is having a renaissance in recent years owing to its potential to address the incompletely understood complexity of diseases. Herein, the C. elegans PDD model was established to pave the way for the future phenotypic discovery of potential agents for treating PQ poisoning. METHODS: C. elegans were treated with PQ-containing solid medium followed by statistical analysis of worm survival, pharyngeal pumping, and movement ability. Furthermore, coenzyme Q10 (CoQ10) was used to test the C. elegans model of PQ poisoning by measuring the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), mitochondrial morphology, and worm survival rate. Additionally, we used the classic mice model of PQ intoxication to evaluate the validity of the C. elegans model of PQ poisoning by measuring the effect of CoQ10 as a potential antidote for PQ poisoning. RESULTS: In the C. elegans model of PQ poisoning, 5 mg/mL PQ increased the levels of ROS, MDA content, mitochondrial fragments, which significantly shortened the lifespan, while CoQ10 alleviated these phenotypes. In the mice model of PQ poisoning, CoQ10 increased the chance of survival in PQ poisoned mice while reducing ROS, MDA content in lung tissue and inhibiting PQ-induced lung edema. Moreover, CoQ10 alleviated the lung morphopathological changes induced by PQ. CONCLUSION: Here we established a C. elegans model of PQ poisoning, whose validity was confirmed by the classic mice model of PQ intoxication.