Targeting thalamic circuits rescues motor and mood deficits in PD mice.

Although bradykinesia, tremor and rigidity are the hallmark motor defects in patients with Parkinson's disease (PD), patients also experience motor learning impairments and non-motor symptoms such as depression1. The neural circuit basis for these different symptoms of PD are not well understood. Although current treatments are effective for locomotion deficits in PD2,3, therapeutic strategies targeting motor learning deficits and non-motor symptoms are lacking4-6. Here we found that distinct parafascicular (PF) thalamic subpopulations project to caudate putamen (CPu), subthalamic nucleus (STN) and nucleus accumbens (NAc). Whereas PF→CPu and PF→STN circuits are critical for locomotion and motor learning, respectively, inhibition of the PF→NAc circuit induced a depression-like state. Whereas chemogenetically manipulating CPu-projecting PF neurons led to a long-term restoration of locomotion, optogenetic long-term potentiation (LTP) at PF→STN synapses restored motor learning behaviour in an acute mouse model of PD. Furthermore, activation of NAc-projecting PF neurons rescued depression-like phenotypes. Further, we identified nicotinic acetylcholine receptors capable of modulating PF circuits to rescue different PD phenotypes. Thus, targeting PF thalamic circuits may be an effective strategy for treating motor and non-motor deficits in PD.

"Self-inactivating" rabies viruses are susceptible to loss of their intended attenuating modification.

Monosynaptic tracing using rabies virus is an important technique in neuroscience, allowing brain-wide labeling of neurons directly presynaptic to a targeted neuronal population. A 2017 article reported the development of a noncytotoxic version-a major advance-based on attenuating the rabies virus by the addition of a destabilization domain to the C terminus of a viral protein. However, this modification did not appear to hinder the ability of the virus to spread between neurons. We analyzed two viruses provided by the authors and show here that both were mutants that had lost the intended modification, explaining the paper's paradoxical results. We then made a virus that actually did have the intended modification in at least the majority of virions and found that it did not spread efficiently under the conditions described in the original paper, namely, without an exogenous protease being expressed in order to remove the destabilization domain. We found that it did spread when the protease was supplied, although this also appeared to result in the deaths of most source cells by 3 wk postinjection. We conclude that the new approach is not robust but that it could become a viable technique given further optimization and validation.

Anterior thalamic circuits crucial for working memory.

Alterations in the structure and functional connectivity of anterior thalamic nuclei (ATN) have been linked to reduced cognition during aging. However, ATN circuits that contribute to higher cognitive functions remain understudied. We found that the anteroventral (AV) subdivision of ATN is necessary specifically during the maintenance phase of a spatial working memory task. This function engages the AV→parasubiculum (PaS)→entorhinal cortex (EC) circuit. Aged mice showed a deficit in spatial working memory, which was associated with a decrease in the excitability of AV neurons. Activation of AV neurons or the AV→PaS circuit in aged mice was sufficient to rescue their working memory performance. Furthermore, rescued aged mice showed improved behavior-induced neuronal activity in prefrontal cortex (PFC), a critical site for working memory processes. Although the direct activation of PFC neurons in aged mice also rescued their working memory performance, we found that these animals exhibited increased levels of anxiety, which was not the case for AV→PaS circuit manipulations in aged mice. These results suggest that targeting AV thalamus in aging may not only be beneficial for cognitive functions but that this approach may have fewer unintended effects compared to direct PFC manipulations.

MFN2 regulates progesterone biosynthesis and proliferation of granulosa cells during follicle selection in hens.

Follicle selection in hens refers to a biological process that only one small yellow follicle (SYF) is selected daily or near-daily for following hierarchical development (from F5/F6 to F1) until ovulation. MFN2 is a kind of GTPases located on the mitochondrial outer membrane, which plays a crucial role in mitochondrial fusion. This study aimed to elucidate the role of MFN2 in proliferation and progesterone biosynthesis of granulosa cells (GCs) during follicle selection in hens. The results showed that GCs began to produce progesterone (P4) after follicle selection, accompanied with changes from multi-layer with flat cells to single layer with cubic cells. MFN2 was detected in GCs of follicles from SYF to F1. After follicle selection, the expression level of MFN2 in GCs upregulated significantly, accompanied with increases in P4 biosynthesis, ATP production, mitochondrial DNA (mtDNA) copy numbers of granulosa cells. FSH (80 ng/mL) facilitated the effects of P4 biosynthesis and secretion, ATP production, mtDNA copy numbers, cell proliferation and the MFN2 transcription of granulosa cells from F5 (F5G) in vitro. However, FSH treatment did not promote P4 secretion in granulosa cells from SYF (SYFG) in vitro. Meanwhile, we observed that change fold of MFN2 transcription, ATP production, mtDNA copy numbers and cell proliferation rate in F5G after treatment with FSH were greater than those in SYFG. Furthermore, expression levels of MFN2 protein and messenger RNA in F5G were significantly higher than those in SYFG after treatment with FSH. P4 biosynthesis, ATP production, mtDNA copy numbers as well as cell proliferation reduced significantly in F5G with MFN2 knockdown. Oppositely, P4 biosynthesis, ATP production, mtDNA copy numbers and cell proliferation increased significantly in SYFG after the overexpression of MFN2. Our results suggest that the upregulation of MFN2 may be involved in the initiation of P4 biosynthesis, and promotion of GCs proliferation during follicle selection.

Self-Illuminating Copper-Luminol Coordination Polymers for Bioluminescence Imaging of Oxidative Damage.

Timely detection of reactive oxygen species (ROS) accumulated during inflammation is essential for an early disease diagnosis. Compared to fluorescence probes with limited sensitivity and accuracy, chemiluminescence (CL) imaging offers the potential for highly sensitive molecular visualization of ROS by minimizing background interferences. However, the development of bright and easily manufacturable CL probes for ROS imaging remains challenging. In this study, a novel chemiluminescent nanoprobe named Cu-Lum@NPs for ROS imaging in inflammation was synthesized by using a one-step solvothermal method. The Cu-Lum@NPs, which are composed of coordination polymers containing copper ions and luminol (Lum), demonstrate intrinsic peroxidase-like activity that relies on Cu(I) as the catalytic active center to initiate the Fenton reaction. This catalytic process facilitates the decomposition of hydrogen peroxide (H2O2) into hydroxyl radicals (•OH) and superoxide anion radicals (O2•-), leading to the oxidation of Lum and inducing strong luminescence. Cu-Lum@NPs, displaying nanozyme characteristics, were observed to accelerate and enhance the ROS-responsive luminescence (10-1600-fold in solution and over 100-fold in neutrophils) and notably extend persistent luminescence. The Cu-Lum@NPs allowed for CL imaging of endogenous ROS in living cells and animals with an outstanding signal-to-noise ratio exceeding 96 and facilitated oxidative damage luminescence imaging for tissue-specific detection. The study presents Cu-Lum@NPs, a highly sensitive and easily manufacturable chemiluminescent nanoprobe for ROS imaging both in vitro and in vivo, exhibiting enhanced luminescence and prolonged persistence for ROS-related disease detection.

Densovirus infection facilitates plant-virus transmission by an aphid.

The interactions among plant viruses, insect vectors, and host plants have been well studied; however, the roles of insect viruses in this system have largely been neglected. We investigated the effects of MpnDV infection on aphid and PVY transmission using bioassays, RNA interference (RNAi), and GC-MS methods and green peach aphid (Myzus persicae (Sulzer)), potato virus Y (PVY), and densovirus (Myzus persicae nicotianae densovirus, MpnDV) as model systems. MpnDV increased the activities of its host, promoting population dispersal and leading to significant proliferation in tobacco plants by significantly enhancing the titer of the sesquiterpene (E)-ß-farnesene (EßF) via up-regulation of expression levels of the MpFPPS1 gene. The proliferation and dispersal of MpnDV-positive individuals were faster than that of MpnDV-negative individuals in PVY-infected tobacco plants, which promoted the transmission of PVY. These results combined showed that an insect virus may facilitate the transmission of a plant virus by enhancing the locomotor activity and population proliferation of insect vectors. These findings provide novel opportunities for controlling insect vectors and plant viruses, which can be used in the development of novel management strategies.

Circadian misalignment impairs oligodendrocyte myelination via Bmal1 overexpression leading to anxiety and depression-like behaviors.

Circadian misalignment (CM) caused by shift work can increase the risk of mood impairment. However, the pathological mechanisms underlying these deficits remain unclear. In the present study, we used long-term variable photoperiod (L-VP) in wild-type mice to better simulate real-life shift patterns and study its effects on the prefrontal cortex (PFC) and hippocampus, which are closely related to mood function. The results showed that exposure to L-VP altered the activity/rest rhythms of mice, by eliciting phase delay and decreased amplitude of the rhythms. Mice with CM developed anxiety and depression-like manifestations and the number of mature oligodendrocytes (OL) was reduced in the medial prefrontal cortex and hippocampal CA1 regions. Mood impairment and OL reduction worsened with increased exposure time to L-VP, while normal photoperiod restoration had no effect. Mechanistically, we identified upregulation of Bmal1 in the PFC and hippocampal regions of CM mice at night, when genes related to mature OL and myelination should be highly expressed. CM mice exhibited significant inhibition of the protein kinase B (AKT)/mTOR signaling pathway, which is directly associated to OL differentiation and maturation. Furthermore, we demonstrated in the OL precursor cell line Oli-Neu that overexpression of Bmal1 inhibits AKT/mTOR pathway and reduces the expression of genes OL differentiation. In conclusion, BMAL1 might play a critical role in CM, providing strong research evidence for BMAL1 as a potential target for CM therapy.

Fuziline Ameliorates Glucose and Lipid Metabolism by Activating Beta Adrenergic Receptors to Stimulate Thermogenesis.

Radix aconiti carmichaeli is a widely used traditional Chinese medicine that has been found to be effective in treating cardiovascular diseases and metabolic disorders. Patients with these diseases often experience a heat generation disorder, which is characterized by chilliness and can worsen the progression of the disease. This study established an in vitro screening model combining the examination of cellular mitochondrial membrane potential and mitochondrial temperature to screen drugs with thermogenic activity. After differentiation and determination of the content of characteristic metabolites of the drug-containing serum blood components, it was found that Fuziline (FZL) is the key thermogenic property in Radix aconiti carmichaeli, responsible for its thermogenic effects with a high relative importance of 33%. Experiments were conducted to evaluate the thermogenic activity of Radix aconiti carmichaeli and FZL in vivo by assessing temperature changes in various organs, including the rectum, liver, and brown adipose tissue. Moreover, the effects of intracellular ß3-adrenergic receptor (ß3-AR) agonistic effects were evaluated using transient ß3-AR transfection and dual-luciferase assay systems. The molecular mechanism by which FZL promotes thermogenesis and improves mitochondrial function was investigated by verifying the ß-adrenergic receptors (ß-AR) downstream signaling pathway. The results suggest that FZL activates ß-AR nonselectively, which in turn activates the downstream cAMP-PKA signaling pathway and leads to an increase in liver glycogenolysis and triglyceride hydrolysis, accompanied by enhancing mitochondrial energy metabolism. Consequently, the liver and brown adipose tissue receive energy to generate heat. In summary, these findings provide insight into the therapeutic application of Radix aconiti carmichaeli for metabolic disorders associated with heat generation disorders.

Mesenchymal stem cells-derived exosomes prevent sepsis-induced myocardial injury by a CircRTN4/miR-497-5p/MG53 pathway.

Sepsis is a life-threatening organ function dysfunction featured by stimulated oxidative stress and inflammatory responses, in which about 40%-60% of sepsis patients are accompanied with cardiac dysfunction. Mesenchymal stem cells (MSCs)-derived exosomes exert critical roles in the treatment of multiple diseases through transferring non-coding RNAs. Circular RNA (circRNA) is a novel form of functional RNAs that involves in the progression of multiple cardiac pathological condition. Nevertheless, the function of MSCs-derived exosomal circRTN4 in sepsis-induced myocardial injury is still obscure. Significantly, FISH assay demonstrated the location of circRTN4 in cytoplasm of cardiomyocytes. The expression of circRTN4 was reduced in the cardiac tissues from caecal ligation and puncture (CLP) rats and LPS-treated cardiomyocytes. CircRTN4 could be delivered to cardiomyocytes cells via MSCs-derived exosomes. The cardiac injury and apoptosis were induced in the CLP rats and the treatment of MSCs-derived exosomal circRTN4 relieved the phenotypes. MSCs-derived exosomal circRTN4 notably suppressed the upregulated ROS level in the CLP rats. The activity of SOD and GSH was repressed in CLP rats, in which MSCs-derived exosomal circRTN4 rescued the activity in the rats. The upregulated IL-1ß, IL-6, and TNF-α levels in CLP rats were reduced by the treatment of MSCs-derived exosomal circRTN4. MSCs-derived exosomal circRTN4 improved cell survival and suppressed apoptosis of LPS-treated cardiomyocytes. CircRTN4 direct interact with miR-497-5p to upregulate MG53 expression in cardiomyocytes. MSCs-derived exosomal circRTN4 relieves LPS-stimulated cardiomyocyte damage via targeting miR-497-5p/MG53 axis. Therefore, we determine that MSCs-derived exosomes prevent sepsis-induced myocardial injury by a circRTN4/miR-497-5p/MG53 pathway. Our data provides novel insight into the regulatory mechanism by which MSCs-derived exosomal circRTN4 regulates sepsis-induced myocardial injury. MSCs-derived exosomal circRTN4 may be applied as a promising therapeutic approach for sepsis-induced myocardial injury.

Harm of circadian misalignment to the hearts of the adolescent wistar rats.

PURPOSE: The purpose of this study was to observe the harm of circadian misalignment (CM), caused by an inverted photoperiod (IP), on the hearts of the adolescent Wistar rats, and to explore the mechanisms leading to harm. METHODS: An IP was used to create a CM model. A total of 174 Wistar rats were randomly divided into circadian alignment (CA) and CM groups (87 rats per group). The different activity rhythms of the two groups of rats were adjusted through different light/dark cycles for 90 days. We recorded the rhythmic activity trajectory and sleep time of the rats. After 90 days of modeling, we performed various analyses (i.e., blood pressure, weight, cardiac ultrasound tests, serological tests, cardiac tissue immunofluorescence, immunohistochemistry, transmission electron microscopy on myocardial mitochondria, western blotting, and quantitative polymerase chain reactions). RESULTS: (1) The IP protocol caused CM in rats. (2) CM rats showed significantly higher blood pressure during the day (resting phase). They also showed significantly higher serum levels of angiotensin II and epinephrine during the day compared to the CA rats. (3) CM caused up-regulation of gene expression of adrenergic receptors α1 (α1-AR) and ß1 (ß1-AR) and down-regulation of the glucocorticoid receptor (Gr) gene expression in rat hearts. It also caused downregulation of Bmal1 expression. In addition, the changes in Bmal1 and Per2 correlated with the changes in ß1-AR and α1-AR. (4) CM had adverse effects on multiple molecular proteins of the heart. (5) CM increased the collagen fibers in the rat heart and increased the destruction of mitochondria. (6) Eventually, CM caused a decrease in the pumping function of the heart and decreased the coronary blood flow rate. CONCLUSIONS: (1) CM significantly affected the cardiac structure and function in the adolescent rats through a variety of mechanisms. (2) CM can regulate the expression of myocardial clock genes, and it is likely to have an impact on the heart through this pathway.

Novel natural inhibitors targeting KRAS G12C by computational study.

Ideal leading and nominee compounds with inhibiting effects on KRAS G12C were selected from the ZINC database, laying a cornerstone for the progress of anticancer drugs. A variety of computational virtual screening methods were utilized to screen possible inhibitors of KRAS G12C. LibDock was utilized to estimate 17 930 compounds and the top 20 were nominated for additional study, which was absorption, distribution, metabolism, and excretion and harmfulness prediction. Molecule docking was employed to prove the binding connection between certain ligands and KRAS G12C. Natural novel compounds ZINC000012494057 and ZINC000003789195 were selected to bind stably with KRAS G12C. In addition, they had lower scores in Ames mutagenicity, rodent carcinogenicity, cytochrome P450 2D6(CYP2D6) tolerance, and non-developmental toxicity potential. Molecular dynamic simulations demonstrate that the combination of ZINC000012494057 and ZINC000003789195 with KRAS G12C has more favorable potential energy, which provides conditions for their stable existence in the natural environment. Natural compounds ZINC000012494057 and ZINC000003789195 were identified as KRAS G12C potential inhibitors. These two compounds have been verified to have enormous importance for the progress of anticancer medicines.

Okicamelliaside targets the N-terminal chaperone pocket of HSP90 disrupts the chaperone protein interaction of HSP90-CDC37 and exerts antitumor activity.

Heat shock protein 90 (HSP90) has been recognized as a crucial target in cancer cells. However, various toxic reactions targeting the ATP binding site of HSP90 may not be the best choice for HSP90 inhibitors. In this paper, an ellagic acid derivative, namely, okicamelliaside (OCS), with antitumor effects was found. To identify potential anti-cancer mechanisms, an OCS photosensitive probe was applied to target fishing and tracing. Chemical proteomics and protein-drug interaction experiments have shown that HSP90 is a key target for OCS, with a strong binding affinity (KD = 6.45 µM). Mutation analysis of the target protein and molecular dynamics simulation revealed that OCS could competitively act on the key Glu-47 site at the N-terminal chaperone pocket of HSP90, where the co-chaperone CDC37 binds to HSP90, affect its stability and reduce the ∆Gbind of HSP90-CDC37. It was demonstrated that OCS destroys the protein-protein interactions of HSP90-CDC37; selectively affects downstream kinase client proteins of HSP90, including CDK4, P-AKT473, and P-ERK1/2; and exerts antitumor effects on A549 cells. Furthermore, tumor xenograft experiments demonstrated high antitumor activity and low toxicity of OCS in the same way. Our findings identified a novel N-terminal chaperone pocket natural inhibitor of HSP90, that is, OCS, which selectively inhibits the formation of the HSP90-CDC37 protein complex, and provided further insight into HSP90 inhibitors for anti-cancer candidate drugs.

Postpartum Levothyroxine Adjustment and Its Impact Factors in Women With Hypothyroidism in Pregnancy.

OBJECTIVE: Women with hypothyroidism need to increase exogenous thyroid hormone levels during pregnancy to reduce adverse outcomes. Few studies have reported the effect of gestational levothyroxine (LT4) variations on postpartum LT4 treatment. METHODS: Women were classified as having subclinical hypothyroidism (SCH) (n = 101), overt hypothyroidism (OH) caused by autoimmune thyroiditis (AIT-OH), OH following thyroidectomy for benign thyroid disease (BA-OH) (n = 66), and OH after surgery for papillary thyroid cancer (PTC-OH) (n = 46). Thyroid function was monitored, and LT4 therapy was adjusted accordingly. RESULTS: After delivery, all women with SCH stopped LT4 treatment, and 57.4% of them restarted LT4 treatment in the following 1 year, independently of the gestational LT4 variations. Among patients with OH, after adjusted by gestational body weight, 49.1% of them had LT4 doses less than the prepregnancy dose (baseline) in late pregnancy, leading to LT4 reduction in postpartum. The LT4 dose was reduced to approximately 50% baseline for women with AIT-OH and BA-OH and reduced by 27% for women with PTC-OH. The reduction reasons for AIT-OH and BA-OH were thyroid-stimulating hormone levels of <2.5 mU/L during pregnancy and postpartum thyrotoxicosis occurrence (39.4%), and for PTC-OH, the reason was thyroid-stimulating hormone overinhibition (<1.0 mU/L) before delivery. CONCLUSION: For patients with SCH, postpartum LT4 treatment could initially be suspended. For women with OH, if the LT4 dose in late pregnancy was less than baseline, a prepregnancy dose reduced by 50%, 50%, and 27% should be applied after delivery for women with AIT-OH, BA-OH, and PTC-OH, respectively.

Intestinal flora variation reflects the short-term damage of microplastic to the intestinal tract in mice.

The potential toxicity of microplastic (MPs) to organisms has attracted extensive attention. However, due to the subacute toxicity of MPs, the biological effect is hard to verify in short-term exposure experiment. Here, by tracking the dynamics of gut microbes, mice model was utilized to evaluate the toxicity of compositional MPs (PE, PET, PP, PS and PVC). After 7 days digestive exposure, the physiological indicators were normal as the control group that the body weight and serum cholesterol levels were insignificant change. Whereas, through histopathological examination, all the treatment groups suffered colon tissue damage, among which PS had the most inflammatory cells. Moreover, the high-throughput sequencing results revealed great variation of intestinal flora in treated mice. The ratio of Bacteroidetes and Firmicutes in PE, PET and PP treatment groups heighten, and the relative abundance of Ruminococcaceae and Lachnospiraceae increased significantly at family levels. At the genus level, Alistipes bacteria in PS treatment group significantly decreased that is associated with obesity risk. It indicated that MPs induced inflammatory response would further interfere the dynamics of intestinal flora causing health effect in living organisms. This work shed light on MPs toxicity in short-term exposure and supplied research paradigm of MPs health risk assessment.

Accurate Physical Property Predictions via Deep Learning.

Neural networks and deep learning have been successfully applied to tackle problems in drug discovery with increasing accuracy over time. There are still many challenges and opportunities to improve molecular property predictions with satisfactory accuracy even further. Here, we proposed a deep-learning architecture model, namely Bidirectional long short-term memory with Channel and Spatial Attention network (BCSA), of which the training process is fully data-driven and end to end. It is based on data augmentation and SMILES tokenization technology without relying on auxiliary knowledge, such as complex spatial structure. In addition, our model takes the advantages of the long- and short-term memory network (LSTM) in sequence processing. The embedded channel and spatial attention modules in turn specifically identify the prime factors in the SMILES sequence for predicting properties. The model was further improved by Bayesian optimization. In this work, we demonstrate that the trained BSCA model is capable of predicting aqueous solubility. Furthermore, our proposed method shows noticeable superiorities and competitiveness in predicting oil-water partition coefficient, when compared with state-of-the-art graphs models, including graph convoluted network (GCN), message-passing neural network (MPNN), and AttentiveFP.

Genome-wide identification of heat shock transcription factors and potential role in regulation of antioxidant response under hot water and glycine betaine treatments in cold-stored peaches.

BACKGROUND: Heat shock transcription factors (Hsfs) play pivotal roles in plant responses to stress. Although glycine betaine (GB) and hot water (HW) treatments are effective in reducing chilling injury (CI), little is known about the characterization of the Hsfs gene family and its potential roles in alleviating CI by regulating antioxidant systems in peach fruit. RESULTS: In this study, 17 PpHsfs were identified in the peach genome and were investigated using bioinformatics, including chromosomal locations, phylogenetic relationships, gene structure, motifs, and promoter analyses. The expression patterns of PpHsfs under GB and HW treatments were also investigated. The PpHsfs showed different expression patterns in GB- and HW-treated fruit, and most of them were significantly up-regulated by both treatments, especially PpHsfA1a/b, PpHsfA2a, PpHsfA9a, and PpHsfB2a/b. Meanwhile, GB and HW treatments induced higher levels of gene expression and antioxidant enzyme activity of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) compared to the control, contributing to the inhibition of hydrogen peroxide (H2 O2 ) accumulation and superoxide anion (O2 .- ) production. Moreover, the correlation analysis between PpHsfs and antioxidant-related genes showed that three PpAPXs were significantly correlated with ten PpHsfs, whereas PpCAT and PpSOD had no significant correlations with PpHsfs, which indicated that PpAPX might be regulated by PpHsfs. CONCLUSIONS: The results indicated that GB and HW treatments induced different PpHsfs transcript levels to regulate the antioxidant gene expressions, which might be beneficial in inhibiting the accumulation of reactive oxygen species and protecting the integrity of cell structure, thus alleviating the development of CI in peach fruit during cold storage. © 2021 Society of Chemical Industry.

Small-Molecule Fluorogenic Probe for the Detection of Mitochondrial Temperature In Vivo.

Mitochondria, as energy factories, participate in many metabolic processes and play vital roles in cell life. Most human diseases are caused by mitochondrial dysfunction, and mitochondrial temperature is an important indicator of mitochondrial function. Despite the biological importance of mitochondria, there are few tools for detecting changes in mitochondrial temperature in living organisms. Here, we report on a thermosensitive rhodamine B (RhB)-derived fluorogenic probe (RhBIV) that enables fluorescent labeling of cell mitochondria at concentrations as low as 1 µM. We demonstrate that this probe exhibits a temperature-dependent response in cell mitochondria. Furthermore, in mice, it has a long half-life (t1/2) and is primarily enriched in the liver. This unique thermosensitive probe offers a simple, nondestructive method for longitudinal monitoring of mitochondrial temperature both in vitro and in vivo to elucidate fundamental physiological and pathological processes related to mitochondrial function.

Ursolic acid reduces hepatocellular apoptosis and alleviates alcohol-induced liver injury via irreversible inhibition of CASP3 in vivo.

Alcoholic liver disease (ALD) is one of the pathogenic factors of chronic liver disease with the highest clinical morbidity worldwide. Ursolic acid (UA), a pentacyclic terpenoid carboxylic acid, has shown many health benefits including antioxidative, anti-inflammatory, anticancer, and hepatoprotective activities. We previously found that UA was metabolized in vivo into epoxy-modified UA containing an epoxy electrophilic group and had the potential to react with nucleophilic groups. In this study we prepared an alkynyl-modified UA (AM-UA) probe for tracing and capturing the target protein of UA from liver in mice, then investigated the mode by which UA bound to its target in vivo. By conducting proteome identification and bioinformatics analysis, we identified caspase-3 (CASP3) as the primary target protein of UA associated with liver protection. Molecule docking analysis showed that the epoxy group of the UA metabolite reacted with Cys-163 of CASP3, forming a covalent bond with CASP3. The binding mode of the UA metabolites (UA, CM-UA, and EM-UA) was verified by biochemical evaluation, demonstrating that the epoxy group produced by metabolism played an important role in the inhibition of CASP3. In alcohol-treated HepG2 cells, pretreatment with the UA metabolite (10 µM) irreversibly inhibited CASP3 activities, and subsequently decreased the cleavage of PARP and cell apoptosis. Finally, pre-administration of UA (20-80 mg· kg-1 per day, ig, for 1 week) dose-dependently alleviated alcohol-induced liver injury in mice mainly via the inhibition of CASP3. In conclusion, this study demonstrates that UA is a valuable lead compound for the treatment of ALD.

Micelles self-assembled by 3-O-ß-D-glucopyranosyl latycodigenin enhance cell membrane permeability, promote antibiotic pulmonary targeting and improve anti-infective efficacy.

BACKGROUND: Nanoparticle-based pulmonary drug delivery systems are commonly developed and applied for drug-targeted delivery. They exhibit significant advantages compared to traditional pulmonary drug delivery systems. However, developing the formulation of each drug is a time-consuming and laborious task. RESULTS: In this study, a universal lung-targeting nanoparticle was designed and constructed. The self-assembled micelles were composed of a platycodon secondary saponin, 3-O-ß-D-glucopyranosyl platycodigenin 682 (GP-682), based on its specific amphiphilic structure. The GP-682 micelles exhibited a relatively stable zeta potential with a particle size between 60 and 90 nm, and the critical micelle concentration (CMC) value was approximately 42.3 µg/mL. Preincubation of GP-682 micelles markedly enhanced their cell membrane permeability and improved drug uptake in vitro. The results were visualized using fluorescent dye tracing, transmission electron microscopy (TEM) observations and the lactate dehydrogenase (LDH) release assay. The obtained benefits enhanced the distribution of levofloxacin (Lev) in mouse lung tissue and reduced antibiotics overdosing. The acute lung injury mouse model induced by the Pseudomonas aeruginosa PA 14 strain demonstrated that preinjection of GP-682 micelles before antibiotic administration resulted in a higher survival rate and anti-infective efficacy in vivo. It also caused reductions in pulmonary injury, bacterial invasion and cytokine expression compared with treatment with Lev alone. CONCLUSIONS: GP-682 micelles are another nanoparticle-based pulmonary drug delivery system and provide a new lung-targeting therapy option.

Quantitative Proteomics Combined with Affinity MS Revealed the Molecular Mechanism of Ginsenoside Antitumor Effects.

Ginsenosides have previously been demonstrated to effectively inhibit cancer cell growth and survival in both animal models and cell lines. However, the specific ginsenoside component that is the active ingredient for cancer treatment through interaction with a target protein remains unknown. By an integrated quantitative proteomics approach via affinity mass spectrum (MS) technology, we deciphered the core structure of the ginsenoside active ingredient derived from crude extracts of ginsenosides and progressed toward identifying the target protein that mediates its anticancer activity. The Tandem Mass Tag (TMT) labeling quantitative proteomics technique acquired 55620 MS/MS spectra that identified 5499 proteins and 3045 modified proteins. Of these identified proteins, 224 differentially expressed proteins and modified proteins were significantly altered in nonsmall cell lung cancer cell lines. Bioinformatics tools for comprehensive analysis revealed that the Ras protein played a general regulatory role in many functional pathways and was probably the direct target protein of a compound in ginsenosides. Then, affinity MS screening based on the Ras protein identified 20(s)-protopanaxadiol, 20(s)-Ginsenoside Rh2, and 20(s)-Ginsenoside Rg3 had affinity with Ras protein under different conditions. In particular, 20(s)-protopanaxadiol, whose derivatives are the reported antitumor compounds 20(s)-Ginsenoside Rh2 and 20(s)-Ginsenoside Rg3 that have a higher affinity for Ras via a low KD of 1.22 µM and the mutation sites of G12 and G60, was demonstrated to play a core role in those interactions. Moreover, the molecular mechanism and bioactivity assessment results confirmed the identity of the chemical ligand that was directly acting on the GTP binding pocket of Ras and shown to be effective in cancer cell bioactivity profiles.