Mechanism-based inactivation of cytochrome P450 3A by evodol.

Evodol is one of the furanoids isolated from the fruits of Evodia rutaecarpa that has been widely prescribed for the treatment of gastrointestinal diseases in China. The aim of this study was to investigate the inhibitory effect of evodol on CYP3A.A 30-min preincubation of evodol with human liver microsomes raised an obvious left IC50 shift, 3.9-fold for midazolam 1'-hydroxylation and 3.2-fold for testosterone 6ß-hydroxylation. Evodol inactivated CYP3A in a time-, concentration- and NADPH-dependent manner, with KI and kinact of 5.1 µM and 0.028 min-1 for midazolam 1'-hydroxylation and 3.0 µM and 0.022 min-1 for testosterone 6ß-hydroxylation.Co-incubation of ketoconazole attenuated the inactivation while the inclusion of glutathione (GSH) and catalase/superoxide dismutase displayed no such protection.cis-Butene-1, 4-dial (BDA) intermediate derived from evodol were trapped by glutathione and N-acetyl-lysine in microsomes and characterised by HR-MS spectra. The BDA intermediate was believed to play a key role in CYP3A inactivation. CYP3A4 and 2C9 were the primary enzymes contributing to the bioactivation of evodol.To sum up, for the first time evodol was characterised as a mechanism-based inactivator of CYP3A.

[Mechanism of miRNA-3679 Inhibiting Downstream ZADH2-Target Genes to Promote Hepatocellular Carcinoma Cell Proliferation].

Objective: To examine the relationship between miRNA-3679 and hepatocellular carcinoma (HCC) cell lines, and to verify the downstream target genes of miRNA-3679. Methods: PCR was used to determine the expression of miRNA-3679 in liver cancer cell lines, and databases, including ENCORI, miRDB and TargetScan, were used to predict the downstream target genes of miRNA-3679. qPCR of the normal control group (or NC group), miR-3679 inhibitor group and transfection negative control group (or inhibitor NC group) was done to determine the transfection efficiency of the target gene, thereby identifying zinc-binding alcohol dehydrogenase domain containing 2 (ZADH2) as the target gene. Western blot was used to determine the ZADH2 protein expression after miRNA-3679 inhibitor transfection. 5-Ethynyl-2'-deoxyuridine (EdU) staining was done to determine the effect of transfection of miRNA-3679 inhibitor and simultaneous transfection of miRNA-3679 and ZADH2 inhibitors on cell proliferation. Clone formation assay was done to determine the ability of cell clone formation. Flow cytometry was done to examine cell apoptosis. Results: The expression level of miRNA-3679 in HCC cell lines was higher than that in normal human liver cell lines (P<0.05). Through screening conducted with the databases, six genes, including GLUD1, B3GAT1, SLC46A3, MAP2K3, ATF5, and ZADH2, were found to be down-regulated in HCC. qPCR showed that ZADH2 expression increased significantly after transfection with miRNA-3679 inhibitor (P<0.01) and luciferase activity increased after transfection with miR-3679 inhibitor (P<0.01). Western blot results showed that ZADH2 protein expression of the miR-3679 inhibitor group was higher than that of the NC group (P<0.01). EdU analysis showed that the number of positive cells in the miRNA-3679 inhibitor group was lower than that in the NC group and the Inhibitor NC group (P<0.05). The clone count of the miR-3679 inhibitor+si-ZADH2 group was significantly higher than that of the miR-3679 inhibitor group (P<0.01). Flow cytometry showed that the number of apoptotic cells of the miR-3679 inhibitor+si-ZADH2 group was significantly lower than that of the miR-3679 inhibitor group (P<0.01). Conclusion: miRNA-3679 is significantly highly expressed in HCC cells and miRNA-3679 can directly interact with ZADH2 gene and affect its expression. Moreover, miRNA-3679 promotes the proliferation of HCC cells and inhibits their apoptosis by suppressing ZADH2.

Integration of waking experience through dreams considered in light of individual differences in implicit learning ability.

The present study explored whether individual differences in implicit learning were related to the incorporation of waking events into dreams. Participants (N = 60) took part in a sequence learning task, a measure of implicit learning ability. They were then asked to keep a record of their waking experiences (personally significant events [PSEs]/major concerns), as well as their nightly dreams for a week. Of these, the responses of 51 participants were suitable for further analysis in which participants themselves and three independent judges rated the correlation between waking events and dreams of the same day. Implicit learning ability was found to significantly correlate with the incorporation of PSEs into dreams. The present results may lend support to the Horton and Malinowski autobiographical memory (AM) model, which accounts for the activation of memories in dreams as a reflection of sleep-dependent memory consolidation processes that focusses in particular on the hyperassociative nature of AM during sleep.

Insight into Seasonal Change of Phytochemicals, Antioxidant, and Anti-Aging Activities of Root Bark of Paeonia suffruticosa (Cortex Moutan) Combined with Multivariate Statistical Analysis.

Chemical compositions, antioxidants, and anti-aging activities of Cortex Moutan (CM), from different collection periods and different producing areas, were measured and compared in order to obtain excellent CM extracts. The bioactivities of CM extracts were examined by an in vitro antioxidant method and a UVB irradiated human dermal fibroblast (HDF) model. Phytochemical properties were obtained from ultra-fast liquid chromatography quadrupole time-of-flight mass spectrometry (UFLC-Q-TOF-MS) prior to the multivariate statistical analysis. As for the results, the extracts of Heze CM (HZCM) and Luoyang CM (LYCM) collected in June had better in vitro antioxidant activities, significantly increased the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and reduced the content of malondialdehyde (MDA), compared to other CM extracts. HZCM and LYCM extracts could upregulate the relative expression of SOD and GSH-Px mRNA. The extract of HZCM collected in June could significantly repress the production of matrix metalloproteinase 1 (MMP-1) and improve the production of procollagen type I (PCOL)-I in UVB irradiated HDF. In total, 50 compounds, including 17 monoterpenoids, 19 flavonoids, 13 phenols, and 1 amino acid were identified or tentatively identified in the CM extracts. Gallic acid, p-hydroxybenzoic acid, oxypaeoniflorin, paeoniflorin, 1,2,3,4,6-O-pentagalloyl glucose, and paeonol were predominant compounds in the CM extracts. Taken together, CM collected from April to September had better antioxidant and anti-aging effects for external usage.

Surgical approaches for definitive treatment of hepatic alveolar echinococcosis: results of a survey in 178 patients.

Hepatic alveolar echinococcosis (HAE) is a potentially fatal disease caused by the larval growth of Echinococcus multilocularis. We analysed the clinical data of 178 consecutive HAE patients treated with definitive radical surgery at our institution. According to the surgical approach: group A patients underwent direct radical hepatic resection; group B patients first underwent percutaneous puncture external drainage, followed by radical hepatic resection 2 months later; group C patients underwent a two-step hepatic resection; and group D patients underwent liver transplantation. The baseline characteristics, mortality, postoperative complications and recurrence rates were evaluated. Symptoms were present in 79.8% (142/178) patients. Bi-lobar lesion was found in 34 (19.1%, 34/178) patients, 47.2% (84/178) of whom had ⩾2 lesions each. There were no intraoperative deaths. The postoperative mortality was 2.29% in group A, 8.62% in group D and 0% in groups B and C. The main cause of death was a serious postoperative complication (Clavien-Dindo grades III-V). Patients were followed-up systematically for a median of 35.8 months (8-72) without recurrence. Active HAE should be treated by radical liver resection, and the complicated alveolar echinococcosis of the liver has been managed whenever possible using principles of radical liver resection by experienced hepatic surgeons.

Asperuloside and Asperulosidic Acid Exert an Anti-Inflammatory Effect via Suppression of the NF-κB and MAPK Signaling Pathways in LPS-Induced RAW 264.7 Macrophages.

Hedyotis diffusa is a folk herb that is used for treating inflammation-related diseases in Asia. Previous studies have found that iridoids in H. diffusa play an important role in its anti-inflammatory activity. This study aimed to investigate the anti-inflammatory effect and potential mechanism of five iridoids (asperuloside (ASP), asperulosidic acid (ASPA), desacetyl asperulosidic acid (DAA), scandoside methyl ester (SME), and E-6-O-p-coumaroyl scandoside methyl ester (CSME)) that are presented in H. diffusa using lipopolysaccharide (LPS)-induced RAW 264.7 cells. ASP and ASPA significantly decreased the production of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in parallel with the inhibition of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α, and IL-6 mRNA expression in LPS-induced RAW 264.7 cells. ASP treatment suppressed the phosphorylation of the inhibitors of nuclear factor-kappaB alpha (IκB-α), p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). The inhibitory effect of ASPA was similar to that of ASP, except for p38 phosphorylation. In summary, the anti-inflammatory effects of ASP and ASPA are related to the inhibition of inflammatory cytokines and mediators via suppression of the NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways, which provides scientific evidence for the potential application of H. diffusa.

Scandoside Exerts Anti-Inflammatory Effect Via Suppressing NF-κB and MAPK Signaling Pathways in LPS-Induced RAW 264.7 Macrophages.

The iridoids of Hedyotis diffusa Willd play an important role in the anti-inflammatory process, but the specific iridoid with anti-inflammatory effect and its mechanism has not be thoroughly studied. An iridoid compound named scandoside (SCA) was isolated from H. diffusa and its anti-inflammatory effect was investigated in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. Its anti-inflammatory mechanism was confirmed by in intro experiments and molecular docking analyses. As results, SCA significantly decreased the productions of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) and inhibited the levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α and IL-6 messenger RNA (mRNA) expression in LPS-induced RAW 264.7 macrophages. SCA treatment suppressed the phosphorylation of inhibitor of nuclear transcription factor kappa-B alpaha (IκB-α), p38, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). The docking data suggested that SCA had great binding abilities to COX-2, iNOS and IκB. Taken together, the results indicated that the anti-inflammatory effect of SCA is due to inhibition of pro-inflammatory cytokines and mediators via suppressing the nuclear transcription factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways, which provided useful information for its application and development.

The anti-inflammatory effect of Andrographis paniculata (Burm. f.) Nees on pelvic inflammatory disease in rats through down-regulation of the NF-κB pathway.

BACKGROUND: Andrographis paniculata (Burm. f.) Nees (APN), a principal constituent of a famous traditional Chinese medicine Fukeqianjin tablet which is used for the treatment of pelvic inflammatory disease (PID), has been reported to have anti-inflammatory effect in vitro. However, whether it has pharmacological effect on PID in vivo is unclear. Therefore, the aim of this study is to test the anti-inflammatory effect of APN and illuminate a potential mechanism. METHODS: Thirty-six female specific pathogen-free SD rats were randomly divided into control group, PID group, APN1 group, APN2 group, APN3 group and prednisone group. Pathogen-induced PID rats were constructed. The APN1, APN2 and APN3 group rats were orally administrated with APN extract at different levels. The prednisone group rats were administrated with prednisone. Eight days after the first infection, the histological examination of upper genital tract was carried out, and enzyme-linked immunosorbent assay (ELISA) was carried out using homogenate of the uterus and fallopian tube. Furthermore, immunohistochemical evaluations of NF-κB p65 and IκB-α in uterus was conducted. RESULTS: APN obviously suppressed the infiltrations of neutrophils and lymphocytes, and it could significantly reduce the excessive production of cytokines and chemokines including IL-1ß, IL-6, CXCL-1, MCP-1 and RANTES in a dose-dependent manner. Furthermore, APN could block the pathogen-induced activation of NF-κB pathway. CONCLUSION: APN showed potent anti-inflammatory effect on pathogen-induced PID in rats, with a potential mechanism of inhibiting the NF-κB signal pathway.

The Hedyotis diffusa Willd. (Rubiaceae): A Review on Phytochemistry, Pharmacology, Quality Control and Pharmacokinetics.

Hedyotis diffusa Willd (H. diffusa) is a well-known Chinese medicine with a variety of activities, especially its anti-cancer effect in the clinic. Up to now, 171 compounds have been reported from H. diffusa, including 32 iridoids, 26 flavonoids, 24 anthraquinones, 26 phenolics and their derivatives, 50 volatile oils and 13 miscellaneous compounds. In vitro and in vivo studies show these phytochemicals and plant extracts to exhibit a range of pharmacological activities of anti-cancer, antioxidant, anti-inflammatory, anti-fibroblast, immunomodulatory and neuroprotective effects. Although a series of methods have been established for the quality control of H. diffusa, a feasible and reliable approach is still needed in consideration of its botanical origin, collecting time and bioactive effects. Meanwhile, more pharmacokinetics researches are needed to illustrate the characteristics of H. diffusa in vivo. The present review aims to provide up-to-date and comprehensive information on the phytochemistry, pharmacology, quality control and pharmacokinetic characteristics of H. diffusa for its clinical use and further development.

Chemical Analysis of Dietary Constituents in Rosa roxburghii and Rosa sterilis Fruits.

Both Rosa roxburghii and R. sterilis, belonging to the Rosaceae, are endemic species in Guizhou Province, China. The fruits of these two species are mixed-used as functional food in the region. Aiming to elucidate the phytochemical characteristics of R. roxburghii and R. sterilis fruits, the essential oils and constituents in a methanol extract have been analyzed and compared by GC-MS and UFLC/Q-TOF-MS, respectively. As a result, a total of 135 volatile compounds were identified by GC-MS and 91 components were different between R. roxburghii and R. sterilis fruits; a total of 59 compounds in methanol extracts were identified by UFLC/Q-TOF-MS, including 13 organic acids, 12 flavonoids, 11 triterpenes, nine amino acids, five phenylpropanoid derivatives, four condensed tannins, two stilbenes, two benzaldehyde derivatives and one benzoic acid derivative; and nine characteristic compounds were found between R. roxburghii and R. sterilis fruits. This systematic study plays an important role for R. roxburghii and R. sterilis fruits in the product development.

A comparison of whole cell directed evolution approaches in breeding of industrial strain of Saccharomyces cerevisiae.

OBJECTIVE: To reduce the fermentation cost in very high gravity fermentations of ethanol using Saccharomyces cerevisiae, whole cell directed evolution approaches were carried out. RESULTS: The methods used included cell ploidy manipulation, global transcription machinery engineering and genome shuffling. Ethanol production by the four methods was improved compared with the control. Notably, the ethanol yield of a strain constructed by genome shuffling was enhanced by up to 11 % more than the control reaching 120 g ethanol/l in 35 h using a very high gravity fermentation with 300 g glucose/l. CONCLUSION: Genome shuffling can create strains with improved fermentation characteristics in very high gravity fermentations.

Chemical Profiles and Protective Effect of Hedyotis diffusa Willd in Lipopolysaccharide-Induced Renal Inflammation Mice.

Protective effect of Hedyotis diffusa (H. diffusa) Willd against lipopolysaccharide (LPS)-induced renal inflammation was evaluated by the productions of cytokines and chemokine, and the bioactive constituents of H. diffusa were detected by the ultra-fast liquid chromatography-diode array detector-quadrupole-time of flight mass spectrometry (UFLC-DAD-Q-TOF-MS/MS) method. As the results showed, water extract of H. diffusa (equal to 5.0 g/kg body weight) obviously protected renal tissues, significantly suppressed the productions of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, and monocyte chemoattractant protein (MCP)-1, as well as significantly promoted the production of IL-10 in serum and renal tissues. According the chemical profiles of H. diffusa, flavonoids, iridoid glycosides and anthraquinones were greatly detected in serum from H. diffusa extract treatment mice. Two main chemotypes, including eight flavonoids and four iridoid glycosides were found in renal tissues from H. diffusa extract treatment mice. The results demonstrated that water extract of H. diffusa had protective effect on renal inflammation, which possibly resulted from the bioactive constituents consisting of flavonoids, iridoids and anthraquinones.

Identification and pharmacokinetics of multiple potential bioactive constituents after oral administration of radix astragali on cyclophosphamide-induced immunosuppression in Balb/c mice.

Radix Astragali (RA) is one of the commonly-used traditional Chinese medicines (TCMs) with an immunomodulatory effect confirmed in the clinic. In order to better understand the material basis for the therapeutic effects, this study was to investigate the absorbed components and their pharmacokinetic profile after oral administration of RA on cyclophosphamide-induced immunosuppression in Balb/c mice. As a result, 51 compounds in RA extract and 31 prototype compounds with nine metabolites were detected in mice plasma by the ultra-fast liquid chromatography (UFLC)-DAD-Q-TOF-MS/MS method. The pharmacokinetic parameters of five main constituents, including calycosin-7-O-glucoside, ononin, calycosin, formononetin and astragaloside IV, were obtained using HPLC-MS/MS. These results offered useful information for research on the pharmacological mechanism of RA and for its further development.

HPLC/UV analysis of polyacetylenes, phenylpropanoid and pyrrolidine alkaloids in medicinally used Codonopsis species.

INTRODUCTION: Codonopsis Radix is commonly used as a tonic in traditional Chinese medicine. However, there is no suitable method to assess the quality of Codonopsis Radix based on multiple components having potential bioactivities. OBJECTIVE: To establish a HPLC/UV method for simultaneous quantitation of polyacetylenes (lobetyol, lobetyolin, lobetyolinin, cordifolioidyne B), phenylpropanoid (tangshenoside I) and pyrrolidine alkaloids (codonopyrrolidiums A, B) in Codonopsis Radix. METHODS: Large-scale methanol extraction of Codonopsis Radix, followed by chromatographic separation, provided the seven analytes for quantitation standards. Ultrasound-assisted methanol extracts of samples were analysed using reversed phase, gradient elution HPLC monitored at 215 nm. RESULTS: The method developed allowed efficient separation of the seven compounds and the detection and quantitation limits of the seven analytes were 0.10-0.32 µg/mL and 0.35-1.07 µg/mL, respectively. All calibration curves showed good linearities (r>0.9993) within the test ranges. Intraday and interday precisions were good with RSD<2.84%. The recoveries of all analytes ranged from 95.8 to 104.7%. CONCLUSION: HPLC/UV is an efficient and accurate method of analysis for simultaneous quantitation of seven components in Codonopsis Radix.

Hypericum japonicum Thunb. ex Murray: phytochemistry, pharmacology, quality control and pharmacokinetics of an important herbal medicine.

Hypericum japonicum Thunb. ex Murray is mainly distributed throughout Asia, Oceania and North America and is used as an important herbal medicine. H. japonicum contains many valuable secondary metabolites, such as flavonoids, phloroglucinols and xanthones and has hepatoprotective, anti-tumor, antibacterial, antiviral, and antioxidant activities and effects on the cardiovascular system and immunity. Coupled with phytochemical and pharmacological research, a series of analytical methods have been developed to evaluate the quality of H. japonicum based on its bioactive components. A pharmacokinetics study involved the absorption of two main flavonoids of H. japonicum in rats. This review aims to present an up-to-date and comprehensive overview of the phytochemistry, pharmacology, quality control and pharmacokinetics of H. japonicum, which should be useful for the greater development of H. japonicum, especially in the development of new drugs and therapeutics for various diseases.

The Influence of Mindful Parenting on Children's Creative Tendencies: The Chain Mediating Effect of Parent-Child Intimacy and Connectedness to Nature.

(1) Objectives: The aim of this study was to examine the relationship between mindful parenting and children's creative tendencies and to investigate the mediating role of parent-child intimacy and connectedness to nature in the relationship between mindful parenting and children's creative tendencies. (2) Methods: In this cross-sectional study, nearly 800 mothers of children aged 3-6 were enrolled. General sociodemographic data, the Mindfulness in Parenting Questionnaire (MIPQ), the Creativity Assessment Packet (CAP), the Child-Parent Relationship Scale-Short Form (CPRS-SF), and the Connectedness to Nature Index-Parents of Preschool Children (CNI-PPC) were all included in the questionnaire survey. (3) Results: There were significant positive correlations among mindful parenting, parent-child intimacy, connectedness to nature, and children's creative tendencies. Mindful parenting had a positive predictive effect on children's creative tendencies. Parent-child intimacy played a mediating role between mindful parenting and children's creative tendencies. Connectedness to nature played a mediating role between mindful parenting and children's creative tendencies. The correlation between mindful parenting and children's creative tendencies may be impacted by the chain mediation effects of parent-child intimacy and connectedness to nature. (4) Conclusions: By promoting parent-child intimacy and connectedness to nature, and by utilizing the chain mediating effects of both, mindful parenting positively impacted children's creative tendencies.

The effect of combined use of resin infiltration with different bioactive calcium phosphate-based approaches on enamel white spot lesions: An in vitro study.

OBJECTIVES: This in vitro study aimed to evaluate the effect of resin infiltration combined with casein phosphopeptide-amorphous calcium phosphate with fluoride (CPP-ACPF) or bioactive glass (BAG) on the stability of enamel white spot lesions (WSLs) treatment. MATERIALS AND METHODS: Eighty-four enamel blocks were prepared from the buccal surfaces of sound human premolars. All enamel blocks were placed in a demineralisation solution for 3 days to establish the artificial enamel WSLs. Enamel blocks with WSLs were randomly divided into three groups (n = 28 each group): RI/B: one-off resin infiltration followed by twice daily BAG treatment; RI/C: one-off resin infiltration followed by twice daily CPP-ACPF treatment; RI: one-off resin infiltration treatment only (as control) and subjected to pH cycling for 7 days. Surface morphology, elemental analysis, crystal characteristics, surface roughness and microhardness of enamel surfaces were investigated by scanning electron microscopy and energy-dispersive spectrometry observation, X-ray diffraction (XRD), atomic force microscope and Vickers' hardness testing, respectively. RESULTS: Mean values of the surface roughness (mean±standard deviation (nm)) were 24.52±5.07, 27.39±5.87 and 34.36±4.55 for groups RI/B, RI/C and RI respectively (p = 0.003). The calcium to phosphate ratios were 1.32±0.16, 1.22±0.26 and 0.69±0.24 for groups RI/B, RI/C and RI respectively (p < 0.001). XRD revealed apatite formation in all three groups. The mean enamel surface microhardness (kg/mm2) of the groups were 353.93±28.49, 339.00±27.32 and 330.38±22.55 for groups RI/B, RI/C and RI respectively (p = 0.216). CONCLUSIONS: Resin infiltration combined with CPP-ACPF or BAG remineralisation appears to improve the surface properties of WSLs. CLINICAL SIGNIFICANCE: The combination of resin infiltration and CPP-ACPF/BAG remineralisation may be a potential treatment for the management of the WSLs.

The G protein-coupled receptor ADGRG6 maintains mouse growth plate homeostasis through IHH Signaling.

The cartilage growth plate is essential for maintaining skeletal growth; however, the mechanisms governing postnatal growth plate homeostasis are still poorly understood. Using approaches of molecular mouse genetics and spatial transcriptomics applied to formalin-fixed, paraffin-embedded (FFPE) tissues, we show that ADGRG6/GPR126, a cartilage-enriched adhesion G protein-coupled receptor (GPCR), is essential for maintaining slow-cycling resting zone cells, appropriate chondrocyte proliferation and differentiation, and growth plate homeostasis in mice. Constitutive ablation of Adgrg6 in osteochondral progenitor cells with Col2a1Cre leads to a shortened resting zone, formation of cell clusters within the proliferative zone, and an elongated hypertrophic growth plate, marked by limited expression of PTHrP but increased IHH signaling throughout the growth plate. Attenuation of Smoothened (SMO)-dependent hedgehog signaling restored the Adgrg6 deficiency-induced expansion of hypertrophic chondrocytes, confirming that IHH signaling can promote chondrocyte hypertrophy in a PTHrP-independent manner. In contrast, postnatal ablation of Adgrg6 in mature chondrocytes with AcanCreERT2, induced after the formation of the resting zone, does not affect PTHrP expression but causes an overall reduction of growth plate thickness marked by increased cell death specifically in the resting zone cells and a general reduction of chondrocyte proliferation and differentiation. Spatial transcriptomics reveals that ADGRG6 is essential for maintaining chondrocyte homeostasis by regulating osteogenic and catabolic genes in all the zones of the postnatal growth plates, potentially through positive regulation of SOX9 expression. Our findings elucidate the essential role of a cartilage-enriched adhesion GPCR in regulating cell proliferation and hypertrophic differentiation by regulation of PTHrP/IHH signaling, maintenance of slow-cycle resting zone chondrocytes, and safeguarding chondrocyte homeostasis in postnatal mouse growth plates.

The cartilage growth plate is an essential structure for skeletal growth, however, the mechanisms that govern growth plate homeostasis are still poorly understood. In this study, we showed that an adhesion G protein-coupled receptor (GPCR) named ADGRG6 plays an essential role in maintaining the slow-cycling cells in the resting zone of the growth plate and directing appropriate proliferation and differentiation of the growth plate chondrocytes. Using a technique called spatial transcriptomics, we compared the gene expression profiles in control and Adgrg6 mutant growth plates and found that ADGRG6 prevents premature hypertrophic differentiation of the growth plate chondrocytes by negatively regulating Indian Hedgehog (IHH) signaling. In summary, our findings highlighted the essential role of a cartilage-enriched GPCR in maintaining growth plate homeostasis through IHH signaling.

"All-in-one" metal polyphenol network nanocapsules integrated microneedle patches for lipophagy fueled ferroptosis-mediated multimodal therapy.

Ferroptosis-mediated multimodal therapy has emerged as a promising strategy for tumor elimination, with lipid peroxide (LPO) playing a pivotal role. However, the therapeutic efficiency is limited due to insufficient intracellular levels of free fatty acids (FFA), which severely hinder the production of LPO. To address this limitation, we proposed a lipophagy strategy aimed at degrading lipid droplets (LDs) to release FFA, serving as the essential "fuel" for LPO production. In this study, the lipophagy inducer epigallocatechin gallate (EGCG) was self-assembled with reactive oxygen species (ROS)-producer phenethyl isothiocyanate (PEITC) mediated by Fe2+ to form EFP nanocapsules, which were further integrated into microneedle patches to form a "all-in-one" EFP@MNs. The metal-polyphenol network structure of EFP endow it with photothermal therapy capacity. Upon insertion into tumors, the released EFP nanocapsules were demonstrated to induce lipophagy through metabolic disturbance, thereby promoting LPO production and facilitating ferroptosis. When combined with photothermal therapy, this approach significantly remolded the tumor immune microenvironment by driving tumor-associated macrophages toward M1 phenotype and enhancing dendritic cell maturation. Encouragingly, in conjunction with αPD-L1 treatment, the proposed EFP@MNs exhibited remarkable efficacy in tumor ablation. Our study presents a versatile framework for utilizing microneedle patches to power ferroptosis-mediated multimodal therapy.

Development and validation of a rapid and sensitive assay for the determination of anisodamine in 50 µL of beagle dog plasma by LC-MS/MS.

A simple, rapid, high-throughput, and highly sensitive LC-MS/MS was developed to determine anisodamine in a small volume (50 µL) of beagle dog plasma using atropine sulfate as the internal standard. The analyte and internal standard were isolated from 50 µL plasma samples after a one-step protein precipitation using Sirocco 96-well protein precipitation filtration plates. The separation was accomplished on a Hanbon Hedera CN column (100 × 4.6 mm, 5 µm) and the run time was 4 min. A Micromass Quatro Ultima mass spectrometer equipped with an ESI source was operated in the multiple reaction monitoring mode with the precursor-to-product ion transitions m/z 306.0→140.0 (anisodamine) and 290.0→123.9 (atropine) used for quantitation. The method was sensitive with a low LOQ of 0.05 ng/mL, and good linearity in the range 0.05-50 ng/mL for anisodamine (r(2) ≥ 0.995). All the validation data, such as accuracy, intra- and interrun precision, were within the required limits. The method was successfully applied to the pharmacokinetic study of anisodamine hydrochloride injection in beagle dogs.