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.

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.

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.

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.

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.

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.

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.

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.

Cannabidiol protects against Alzheimer's disease in C. elegans via ROS scavenging activity of its phenolic hydroxyl groups.

Recent discoveries have implicated the potential of Cannabidiol (CBD) in the prevention of Alzheimer's disease (AD). However, how CBD affects such neurodegenerative disorders remains unclear. Herein, Caenorhabditis elegans (C. elegans) was used as the model organism to elucidate the mechanism by which CBD ameliorates AD in vivo. CBD was found to alleviate the progression of Aß-induced AD but not tau protein-induced AD or α-syn-induced Parkinson's disease. CBD inhibited the aggregation of Aß in C. elegans. However, CBD failed to prevent the formation of ß-sheet aggregation in vitro. Moreover, CBD was found to scavenge reactive oxygen species (ROS) in vivo without inducing the overexpression of antioxidative genes. In addition, CBD treatment enhanced the worm resistance to oxidative stress, which was independent of the classical transcription factors DAF-16 and SKN-1. These results supported that the in vivo antioxidative activity of CBD was most likely due to its intrinsic antioxidative property. Furthermore, the phenolic hydroxyl groups of CBD were found to be critical for scavenging ROS in vitro and in vivo, alleviating the aggregation of Aß in vivo, and ameliorating Aß-associated neurotoxicity. These studies show that CBD protects against AD in C. elegans via the ROS scavenging activity of its phenolic hydroxyl groups, which provides insight for further structure-activity relationship studies of CBD as an AD therapeutic.

Engineered Exosomes-Based Photothermal Therapy with MRI/CT Imaging Guidance Enhances Anticancer Efficacy through Deep Tumor Nucleus Penetration.

Exosomes, as natural nanovesicles, have become a spotlight in the field of cancer therapy due to their reduced immunogenicity and ability to overcome physiological barriers. However, the tumor targeting ability of exosomes needs to be improved before its actual application. Herein, a multiple targeted engineered exosomes nanoplatform was constructed through rare earth element Gd and Dy-doped and TAT peptide-modified carbon dots (CDs:Gd,Dy-TAT) encapsulated into RGD peptide engineered exosomes (Exo-RGD), which were used to enhance the effect of cancer imaging diagnosis and photothermal therapy. In vitro and in vivo experiments showed that the resulting CDs:Gd,Dy-TAT@Exo-RGD could effectively accumulate at cancer site with an increased concentration owing to the targeting peptides modification and exosomes encapsulation. The tumor therapy effects of mice treated with CDs:Gd,Dy-TAT@Exo-RGD were heightened compared with mice from the CDs:Gd,Dy control group. After intravenous injection of CDs:Gd,Dy-TAT@Exo-RGD into tumor-bearing mice, the temperature of tumors rose to above 50 °C under NIR irradiation and the localized hyperpyrexia induced by CDs could remarkably ablate tumors. The survival rate of the mice was 100% after 60 days. In addition, the CDs:Gd,Dy-TAT@Exo-RGD exhibited higher MRI/CT imaging contrast enhancement of tumor sites than that of CDs:Gd,Dy. Our study identified that engineered exosomes are a powerful tool for encapsulating multiple agents to enhance cancer theranostic efficiency and provide insight into precise personalized nanomedicine.

Nicotine and its metabolite cotinine target MD2 and inhibit TLR4 signaling.

Nicotine is the principal alkaloid of tobacco often manufactured into cigarettes and belongs to a highly addictive class of drugs. Nicotine attenuates the neuroinflammation induced by microglial activation. However, the molecular target(s) underlying anti-inflammatory action of nicotine has not been fully understood. Considering the psychoactive substances morphine, cocaine, and methamphetamine act as xenobiotic-associated molecular patterns and can be specifically sensed by the innate immune receptor Toll-like receptor 4 (TLR4), here we sought to delineate whether nicotine and/or its metabolite cotinine may be recognized by the innate immune system via myeloid differentiation protein 2 (MD2), an accessory protein of TLR4 that is responsible for ligand recognition. MD2-intrinsic fluorescence titrations, surface plasmon resonance, and competitive displacement binding assays with curcumin (MD2 probe) demonstrated that both nicotine and cotinine targeted the lipopolysaccharide (LPS; TLR4 agonist) binding pocket of MD2 with similar affinities. The cellular thermal shift assay indicated that nicotine binding increased, while cotinine binding decreased, MD2 stability. These biophysical binding results were further supported by in silico simulations. In keeping with targeting MD2, both nicotine and cotinine inhibited LPS-induced production of nitric oxide and tumor necrosis factor alpha (TNF-α) and blocked microglial activation. Neither a pan nicotinic acetylcholine receptor (nAChR) inhibitor nor RNAi for nAChRs abolished the suppressive effect of nicotine- and cotinine-induced neuroinflammation. These data indicate that TLR4 inhibition by nicotine and cotinine at the concentrations tested in BV-2 cells is independent of classic neuronal nAChRs and validate that MD2 is a direct target of nicotine and cotinine in the inhibition of innate immunity.

Targeting the transmembrane domain 5 of latent membrane protein 1 using small molecule modulators.

Protein-protein interactions (PPIs) play a critical role in living cells and represent promising targets for the drug discovery and life sciences communities. However, lateral transmembrane PPIs are difficult targets for small-molecule inhibitor development given less structural information is known and fewer ligand discovery methods have been explored compared to soluble proteins. In this study, the interactions of the transmembrane domain 5 (TMD-5) of latent membrane protein 1 (LMP-1) of Epstein-Barr virus (EBV) were disrupted by pentamidine derivatives to curb the committed step of EBV infection. A pentamidine derivative 2 with a 7-atom di-amide linker had the best activity whilst switching the amide regiochemistry in the linker influenced membrane permeability and abolished anti TMD-5 activity. Molecular dynamics simulations were performed to understand the interaction between pentamidine derivatives and TMD-5, and to rationalise the observed structure-activity relationships. This study explicitly demonstrated that the interaction of small molecule with lipid should be considered alongside interaction with the protein target when designing small molecules targeting the PPIs of TMDs. In all, this study provides proof of concept for the rational design of small molecules targeting transmembrane PPIs.

Dissecting Role of Charged Residue from Transmembrane Domain 5 of Latent Membrane Protein 1 via In Silico Simulations and Wet-Lab Experiments.

Charged residues are frequently found in the transmembrane segments of membrane proteins, which reside in the hydrophobic bilayer environment. Charged residues are critical for the function of membrane protein. However, studies of their role in protein oligomerization are limited. By taking the fifth transmembrane domain (TMD5) of latent membrane protein 1 from the Epstein-Barr virus as a prototype model, in silico simulations and wet-lab experiments were performed to investigate how the charged states affect transmembrane domain oligomerization. Molecular dynamics (MD) simulations showed that the D150-protonated TMD5 trimer was stable, whereas unprotonated D150 created bends in the helices which distort the trimeric structure. D150 was mutated to asparagine to mimic the protonated D150 in TMD5, and the MD simulations of different D150N TMD5 trimers supported that the protonation state of D150 was critical for the trimerization of TMD5. In silico mutations found that D150N TMD5 preferred to interact with TMD5 to form the heterotrimer (1 D150N TMD5:2 protonated TMD5s) rather than the heterotrimer (2 D150N TMD5s:1 protonated TMD5). D150R TMD5 interacted with TMD5 to form the heterotrimer (1 D150R TMD5:2 protonated TMD5). These in silico results imply that D150N TMD5 and D150R TMD5 peptides may be probes for disrupting TMD5 trimerization, which was supported by the dominant-negative ToxR assay in bacterial membranes. In all, this study elucidates the role of charged residues at the membrane milieu in membrane protein oligomerization and provides insight into the development of oligomerization-regulating peptides for modulating transmembrane domain lateral interactions.

Velvet Antler Methanol Extracts Ameliorate Parkinson's Disease by Inhibiting Oxidative Stress and Neuroinflammation: From C. elegans to Mice.

Velvet antler is the traditional tonic food or medicine used in East Asia for treating aging-related diseases. Herein, we try to dissect the pharmacology of methanol extracts (MEs) of velvet antler on Parkinson's disease (PD). Caenorhabditis elegans studies showed that MEs decreased the aggregation of α-synuclein and protected oxidative stress-induced DAergic neuron degeneration. In vitro cellular data indicated that MEs suppressed the LPS-induced MAPKs and NF-κB activation, therefore inhibiting overproduction of reactive oxygen species, nitric oxide, tumor necrosis factor-α, and interleukin-6; blocking microglia activation; and protecting DAergic neurons from the microglia-mediated neurotoxicity. In vivo MPTP-induced PD mouse investigations found that MEs prevented MPTP-induced neuron loss in the substantia nigra and improved the behavioral rotating rod performance in MPTP-treated mice by increasing the expression level of tyrosine hydroxylase (TH) and downregulating α-synuclein protein expression. In all, these results demonstrate that MEs ameliorate PD by inhibiting oxidative stress and neuroinflammation.

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.

Exploring the Thermodynamics of 7-Amino Actinomycin D-Induced Single-Stranded DNA Hairpin by Spectroscopic Techniques and Computational Simulations.

NMR studies have indicated that the anti-tumor therapeutic agent actinomycin D (ACTD) can induce seemingly single-stranded DNA (ssDNA) oligomer 5'-CCGTT3GTGG-3' to form a hairpin structure with tandem GT mismatches at the stem region next to a loop of three stacked thymine bases. In an effort to uncover the preference of binding sequence and to elucidate the thermodynamics properties of the binding, a combination of spectroscopic techniques and computational simulation studies was performed with d(CCGTTnGTGG) and d(CCGAAnGAGG) (denoted as GTTn and GAAn, respectively; n = 3, 5, and 7) sequences. In the presence of 7-amino actinomycin D (7AACTD), all the six oligomers formed stable hairpin structures. The GTT5-7AACTD/GAA5-7AACTD hairpin structure was more stable than the corresponding GTTn-7AACTD and GAAn-7AACTD (n = 3, 7). No significant ΔG difference was observed between GTTn-7AACTD and GAAn-7AACTD complexes with the same loop length. In agreement with the 7AACTD-induced hairpin stability results, the binding affinity of GTTn and GAAn with 7AACTD increased from n = 3 to n = 5 and then decreased when n is 7. Moreover, GTTn and GAAn with the same loop length showed comparable binding affinities to 7AACTD. Furthermore, molecular dynamics simulations found that van der Waals interactions between GTTn/GAAn and 7AACTD were the primary attractive forces for 7AACTD binding, and the electrostatic interactions between the carbonyl groups of 7AACTD and bases in the hairpin were the major unfavorable forces. These findings furthered our understanding that 7AACTD is sensitive to the loop size and sequence as well as tandem GT/GA mismatches of their deoxyribonucleic acid (DNA) targets. A deep understanding of the thermodynamics and the molecular recognition mechanism of 7AACTD with ssDNAs would further the development of ACTD-like antitumor agents.

A longitudinal observational retrospective study on risk factors and predictive model of PICC associated thrombosis in cancer patients.

To analyze the incidence of PICC associated venous thrombosis. To predict the risk factors of thrombosis. To validate the best predictive model in predicting PICC associated thrombosis. Consecutive oncology cases in 341 who initially naive intended to be inserted central catheter for chemotherapy, were recruited to our dedicated intravenous lab. All patients used the same gauge catheter, Primary endpoint was thrombosis formation, the secondary endpoint was infusion termination without thrombosis. Two patients were excluded. 339 patients were divided into thrombosis group in 59 (17.4%) and non-thrombosis Group in 280 (82.6%), retrospectively. Tumor, Sex, Age, Weight, Height, BMI, BSA, PS, WBC, BPC, PT, D-dimer, APTT, FIB, Smoking history, Location, Catheter length, Ratio and Number as independent variables were analyzed by Fisher's scoring, then Logistic risk regression, ROC analysis and nomogram was introduced. Total incidence was 17.4%. Venous mural thrombosis in 2 (3.4%), "fibrin sleeves" in 55 (93.2%), mixed thrombus in 2 (3.4%), symptomatic thrombosis in 2 (3.4%), asymptomatic thrombosis in 57 (96.6%), respectively. Height (χ² = 4.48, P = 0.03), D-dimer (χ² = 37.81, P < 0.001), Location (χ² = 7.56, P = 0.006), Number (χ² = 43.64, P < 0.001), Ratio (χ² = 4.38, P = 0.04), and PS (χ² = 58.78, P < 0.001), were statistical differences between the two groups analyzed by Fisher's scoring. Logistic risk regression revealed that Height (ß = -0.05, HR = 0.95, 95%CI: 0.911-0.997, P = 0.038), PS (ß = 1.07, HR = 2.91, 95%CI: 1.98-4.27, P < 0.001), D-dimer (ß0.11, HR = 1.12, 95%CI: 1.045-1.200, P < 0.001), Number (ß = 0.87, HR = 2.38, 95% CI: 1.619-3.512, P < 0.001) was independently associated with PICC associated thrombosis. The best prediction model, D-dimer + Number as a novel co-variable was validated in diagnosing PICC associated thrombosis before PICC. Our research revealed that variables PS, Number, D-dimer and Height were risk factors for PICC associated thrombosis, which were slightly associated with PICC related thrombosis, in which, PS was the relatively strongest independent risk factor of PICC related thrombosis.

Exosomes of Antler Mesenchymal Stem Cells Improve Postoperative Cognitive Dysfunction in Cardiopulmonary Bypass Rats through Inhibiting the TLR2/TLR4 Signaling Pathway.

Postoperative cognitive dysfunction (POCD) is a severe complication of cardiopulmonary bypass (CPB) and has common characteristics such as acute cognitive dysfunction, impaired memory, and inattention. Mesenchymal stem cells (MSCs) are multipotent cells that have therapeutic potentials mainly through paracrine action via secreting growth factors and cytokines. Exosomes are one of the important paracrine factors and have been reported as potential cell-free therapy for the treatment of autoimmune and central nervous system disorders. In this study, we examined exosomes derived from antler MSCs (AMSCs) of POCD rats after CPB and evaluated their potential regulatory mechanisms. AMSC-derived exosomes reduced neurological damage and brain damage and prevent apoptosis in CPB rats. Furthermore, AMSC-derived exosomes were found to reduce hippocampal neuronal apoptosis and the expression of TLR2, TLR4, MyD88, and NF-κB in CPB rats. However, the above effects of AMSC-derived exosomes on CPB rats were abolished partially by toll-like receptor 2/4 (TLR2/TLR4) agonist (LPS-EB). In conclusion, AMSC-derived exosomes can improve cognitive function in CPB rats through inhibiting the TLR2/TLR4 signaling pathway.

Identification of key genes associated with spermatogenesis arrest in fox hybrids using weighted gene co-expression network analysis.

The silver fox and the blue fox represent different genera, but produce viable offspring. Although these hybrids show obvious heterosis, they are completely sterile due to spermatogenic arrest at the early stages of spermatogenesis, especially mitosis and meiosis I; the hybrids produce few spermatogonia and primary spermatocytes, and no secondary spermatocytes. Although the mechanisms of spermatogenic arrest have been well investigated, transcriptomic differences between hybrid and the pure-species testes have not clarified. In the present study, we used RNA sequencing (RNA-Seq) to generate testicular transcriptomic profiles for silver foxes, blue foxes, and reciprocal hybrids during the pre-breeding period and the breeding season. In total, 1,344,022 transcripts (≥200 bp) were generated; 1,057,724 genes were obtained; and 33,423 genes were shown to have fragments per kilobase of transcript per million mapped reads (FPKM) > 0.3. To identify the hub genes associated with spermatogenesis arrest, weighted gene co-expression network analysis (WGCNA) was used. Nine modules were explored. Genes in only a single module were consistently downregulated in the hybrids as compared to the pure species; these genes were significantly associated with fox hybrid male infertility. Six of the genes in this module (CATSPERD, DMRTC2, RNF17, NME5, SPEF2, SPINK2) also play key roles in mitosis and meiosis during spermatogenesis. Therefore, these six genes might be associated with fox hybrid male infertility.