BACKGROUND:During bone remodeling,bone formation and bone resorption are spatially and temporally coordinated,involving intricate interactions between osteoclasts and osteoblasts.Isoginkgetin,a flavonoid found in Ginkgo biloba,has a wide range of anticancer activity and anti-reactive oxygen species activity;however,the effect of isoginkgetin on osteoclast differentiation is unknown.OBJECTIVE:To study the effect and mechanism of action of isoginkgetin on osteoclastogenesis.METHODS:In vitro studies were performed on mouse bone marrow-derived macrophages,and cell counting kit-8 cytotoxicity assay was used to detect the effect of isoginkgetin on cell viability of bone marrow-derived macrophages.Macrophage colony-stimulating factor and receptor activator of nuclear factor kappa-B ligand were used to induce the differentiation of bone marrow-derived macrophages to osteoclasts.Network pharmacology and molecular docking and molecular dynamics simulations were used to predict the processes and targets of the effects of isoginkgetin on the differentiation of osteoclasts.Tartrate-resistant acid phosphatase staining and F-actin staining were used to detect the effects of isoginkgetin on the differentiation and function of osteoclasts.Western blot and RT-PCR were used to detect the effects of isoginkgetin on the expression of genes and proteins related to osteoclast differentiation,reactive oxygen species,and PI3K/AKT pathways.Fluorescent probes were used to detect cellular and mitochondrial reactive oxygen species levels.Flow cytometry technology was used to detect reactive oxygen species levels in cells.RESULTS AND CONCLUSION:(1)Network pharmacology results showed that isoginkgetin affected osteoporosis mainly through the PI3K-AKT pathway and cellular response to drugs and hypoxia,and GSK3β,ESR1,MCL1 and CCNA2 were the key targets.(2)Cell counting kit-8 and tartrate-resistant acid phosphatase staining results showed that isoginkgetin at 8 μmol/L had the most significant inhibitory effect on osteoclastogenesis in vitro,and F-actin results showed that isoginkgetin inhibited osteoclast cytoskeletal actin ring formation in a concentration-dependent manner.(3)Molecular dynamics simulations showed that isoginkgetin bound well to osteoclastogenesis marker proteins(NFATc1,c-Fos,CTSK,and MMP9).Western blot and RT-PCR results indicated that isoginkgetin inhibited the expression of osteoclastogenesis marker proteins and genes(NFATc1,c-Fos,CTSK,and MMP9).(4)Western blot results showed that isoginkgetin inhibited the phosphorylation level of PI3K/AKT/GSK3β and suppressed osteoclastogenesis by activating the PI3K-AKT-GSK3β pathway.(5)The results of reactive oxygen species assay showed that isoginkgetin significantly reduced receptor activator of nuclear factor kappa-B ligand-induced cellular and mitochondrial reactive oxygen species production,and inhibited the differentiation of bone marrow-derived macrophages to osteoclasts.

BACKGROUND:During bone remodeling,bone formation and bone resorption are spatially and temporally coordinated,involving intricate interactions between osteoclasts and osteoblasts.Isoginkgetin,a flavonoid found in Ginkgo biloba,has a wide range of anticancer activity and anti-reactive oxygen species activity;however,the effect of isoginkgetin on osteoclast differentiation is unknown.OBJECTIVE:To study the effect and mechanism of action of isoginkgetin on osteoclastogenesis.METHODS:In vitro studies were performed on mouse bone marrow-derived macrophages,and cell counting kit-8 cytotoxicity assay was used to detect the effect of isoginkgetin on cell viability of bone marrow-derived macrophages.Macrophage colony-stimulating factor and receptor activator of nuclear factor kappa-B ligand were used to induce the differentiation of bone marrow-derived macrophages to osteoclasts.Network pharmacology and molecular docking and molecular dynamics simulations were used to predict the processes and targets of the effects of isoginkgetin on the differentiation of osteoclasts.Tartrate-resistant acid phosphatase staining and F-actin staining were used to detect the effects of isoginkgetin on the differentiation and function of osteoclasts.Western blot and RT-PCR were used to detect the effects of isoginkgetin on the expression of genes and proteins related to osteoclast differentiation,reactive oxygen species,and PI3K/AKT pathways.Fluorescent probes were used to detect cellular and mitochondrial reactive oxygen species levels.Flow cytometry technology was used to detect reactive oxygen species levels in cells.RESULTS AND CONCLUSION:(1)Network pharmacology results showed that isoginkgetin affected osteoporosis mainly through the PI3K-AKT pathway and cellular response to drugs and hypoxia,and GSK3β,ESR1,MCL1 and CCNA2 were the key targets.(2)Cell counting kit-8 and tartrate-resistant acid phosphatase staining results showed that isoginkgetin at 8 μmol/L had the most significant inhibitory effect on osteoclastogenesis in vitro,and F-actin results showed that isoginkgetin inhibited osteoclast cytoskeletal actin ring formation in a concentration-dependent manner.(3)Molecular dynamics simulations showed that isoginkgetin bound well to osteoclastogenesis marker proteins(NFATc1,c-Fos,CTSK,and MMP9).Western blot and RT-PCR results indicated that isoginkgetin inhibited the expression of osteoclastogenesis marker proteins and genes(NFATc1,c-Fos,CTSK,and MMP9).(4)Western blot results showed that isoginkgetin inhibited the phosphorylation level of PI3K/AKT/GSK3β and suppressed osteoclastogenesis by activating the PI3K-AKT-GSK3β pathway.(5)The results of reactive oxygen species assay showed that isoginkgetin significantly reduced receptor activator of nuclear factor kappa-B ligand-induced cellular and mitochondrial reactive oxygen species production,and inhibited the differentiation of bone marrow-derived macrophages to osteoclasts.

Protein structure determines function, and structural information is critical for predicting protein thermostability. This study proposes a novel method for protein thermostability prediction by integrating graph embedding features and network topological features. By constructing residue interaction networks (RINs) to characterize protein structures, we calculated network topological features and utilize deep neural networks (DNN) to mine inherent characteristics. Using DeepWalk and Node2vec algorithms, we obtained node embeddings and extracted graph embedding features through a TopN strategy combined with bidirectional long short-term memory (BiLSTM) networks. Additionally, we introduced the Doc2vec algorithm to replace the Word2vec module in graph embedding algorithms, generating graph embedding feature vector encodings. By employing an attention mechanism to fuse graph embedding features with network topological features, we constructed a high-precision prediction model, achieving 87.85% prediction accuracy on a bacterial protein dataset. Furthermore, we analyzed the differences in the contributions of network topological features in the model and the differences among various graph embedding methods, and found that the combination of DeepWalk features with Doc2vec and all topological features was crucial for the identification of thermostable proteins. This study provides a practical and effective new method for protein thermostability prediction, and at the same time offers theoretical guidance for exploring protein diversity, discovering new thermostable proteins, and the intelligent modification of mesophilic proteins.
Neural Networks, Computer ; Algorithms ; Protein Stability ; Proteins/chemistry* ; Protein Conformation ; Temperature

[This corrects the article DOI: 10.1016/j.apsb.2021.07.004.].

The continuous emergence of SARS-CoV-2 variants as well as other potential future coronavirus has challenged the effectiveness of current COVID-19 vaccines. Therefore, there remains a need for alternative antivirals that target processes less susceptible to mutations, such as the formation of six-helix bundle (6-HB) during the viral fusion step of host cell entry. In this study, a novel high-throughput screening (HTS) assay employing a yeast-two-hybrid (Y2H) system was established to identify inhibitors of HR1/HR2 interaction. The compound IMB-9C, which achieved single-digit micromolar inhibition of SARS-CoV-2 and its Omicron variants with low cytotoxicity, was selected. IMB-9C effectively blocks the HR1/HR2 interaction in vitro and inhibits SARS-CoV-2-S-mediated cell-cell fusion. It binds to both HR1 and HR2 through non-covalent interaction and influences the secondary structure of HR1/HR2 complex. In addition, virtual docking and site-mutagenesis results suggest that amino acid residues A930, I931, K933, T941, and L945 are critical for IMB-9C binding to HR1. Collectively, in this study, we have developed a novel screening method for HR1/HR2 interaction inhibitors and identified IMB-9C as a potential antiviral small molecule against COVID-19 and its variants.

The oxytocin receptor (OXTR) has garnered increasing attention for its role in regulating both mature behaviors and brain development. It has been established that OXTR mediates a range of effects that are region-specific or period-specific. However, the current studies of OXTR expression patterns in mice only provide limited help due to limitations in resolution. Therefore, our objective was to generate a comprehensive, high-resolution spatiotemporal expression map of Oxtr mRNA across the entire developing mouse brain. We applied RNAscope in situ hybridization to investigate the spatiotemporal expression pattern of Oxtr in the brains of male mice at six distinct postnatal developmental stages (P7, P14, P21, P28, P42, P56). We provide detailed descriptions of Oxtr expression patterns in key brain regions, including the cortex, basal forebrain, hippocampus, and amygdaloid complex, with a focus on the precise localization of Oxtr⁺ cells and the variance of expression between different neurons. Furthermore, we identified some neuronal populations with high Oxtr expression levels that have been little studied, including glutamatergic neurons in the ventral dentate gyrus, Vgat⁺Oxtr⁺ cells in the basal forebrain, and GABAergic neurons in layers 4/5 of the cortex. Our study provides a novel perspective for understanding the distribution of Oxtr and encourages further investigations into its functions.
Animals ; Receptors, Oxytocin/metabolism* ; Male ; Brain/growth & development* ; Mice ; Mice, Inbred C57BL ; Neurons/metabolism* ; Single-Cell Analysis ; Gene Expression Regulation, Developmental ; RNA, Messenger/metabolism* ; Animals, Newborn

Objective To investigate the potential beneficial predictors for endovascular treatment(EVT)in case of acute anterior circulation large vessel occlusion with large-core infarction.Methods We made a retrospective inclusion of 92 patients with anterior circulation large vessel occlusion and core infarct volume ranging from 50 to 100 mL,who underwent EVT at Zhangzhou Municipal Hospital of Fujian Province from March 2018 to February 2021.All the patients were confirmed as anterior circulation large infarction and large infarct volume using computed tomography(CT),computed tomography angiography(CTA),and computed tomography perfusion(CTP)before EVT.All clinical and imaging data were collected to observe the postoperative recurrence rate,incidence of symptomatic intracranial hemorrhage(sICH),and functional prognosis 90 days.Then the patients were divided into favorable outcomes group(mRS≤3)and unfavorable outcomes group(mRS＞3)based on Rankin scores 90 days post-stroke.Univariate and multivariate Logistic analysis were conducted to analyze factors influencing the patients'prognosis.By combining the results of multivariate analysis,we constructed receiver operating characteristic(ROC)curves and identified the cut-off value to evaluate the predictive value of age for post-vascular treatment prognosis.Results Among the included patients,the postoperative revascularization rate(mTIICI≥2b)was 95％(88 cases),the rate of favorable outcomes at 90 days postoperatively(mRS≤3)was 32.61％(30 cases),the incidence of sICH was 13％,and the mortality rate was 31.5％.Compared with the unfavorable outcomes group,the favorable outcomes group had a younger age and a higher proportion of males.Multivariate analysis indicated that older age was an independent risk factor for adverse outcomes following endovascular treatment(OR=4.97,95％CI:1.78-13.90,P=0.002).The ROC curve indicated that the area under the curve was maximized at the age of 72.5 years(AUC=0.763,95％CI:0.661-0.864,P＜0.001).Its sensitivity and specificity was 0.565 and 0.833,respectively.Conclusion Age is an independent predictor of the prognosis of acute procirculatory large core stroke.Patients with large core infarction older than 72.5 years may not benefit from endovascular therapy,which needs to be confirmed by a multicenter large sample prospective randomized controlled trial.

Objective To observe the clinical efficacy of Zishen Shujing decoction in the treatment of kidney deficiency and liver depression type premature ovarian failure(POF).Methods A total of 70 female POF patients who visited the Fourth Clinical Medical College of Guangzhou University of Traditional Chinese Medicine from July 2023 to April 2024 were selected as the research subjects,they were divided into traditional Chinese medicine(TCM)treatment group(n=35)and Western medicine treatment group(n=35)by using a random number table method.Western medicine treatment group was treated with femostone,and TCM treatment group was treated with Zishen Shujing decoction.Differences of TCM syndrome scores and clinical efficacy were compared between two groups before and after treatment,and changes of peripheral blood follicle-simulating hormone(FSH),anti-Müllerian hormone(AMH),anal follicle count(AFC)and interleukin(IL)-4,IL-6 before and after treatment were compared.Results The effective rate of TCM treatment group was higher than that of Western medicine treatment group,the difference was statistically significant(P<0.05).The TCM syndrome scores after treatment were lower than before treatment,and TCM treatment group was lower than Western medicine treatment group,the difference was statistically significant(P<0.05).After treatment,FSH levels in two groups were lower than before treatment,AMH and AFC levels were higher than before treatment,and improvement of hormone levels,AMH and AFC in TCM treatment group was better than that in Western medicine treatment group,with statistical significance(P<0.05).After treatment,the anti-inflammatory factors IL-4 were increased,and the pro-inflammatory factors IL-6 were decreased,and the increase effect of anti-inflammatory factors and the decrease effect of pro-inflammatory factors in TCM treatment group were better than those in Western medicine treatment group with statistical significance(P<0.05).Conclusion Zishen Shujing decoction can improve the clinical symptoms,serum hormone levels of POF patients with kidney deficiency and liver depression,improve ovarian function,reduce immune inflammation,reduce oxidative stress,and effectively improve the quality of life of patients.

[Objective]To explore the origin and development relationship,core pathogenesis and medication characteristics of Wendan Decoction,and clarify its application indications.[Methods]Taking Wendan Decoction recorded in YAO Sengyuan's Set of Proven Prescriptions as the main research object,this paper adopts the method of literature search to collect and sort out medical literature related to"insomnia"during the Wei,Jin,Southern and Northern Dynasties,and makes a comparative study of the prescriptions contained therein.It traces the historical origin and the evolution of the main indications of Wendan Decoction,examines contemporary literature related to"gallbladder cold"to explore the theoretical basis of insomnia caused by gallbladder cold,and compares the recorded efficacy of the component herbs in classical materia medica to further analyze its pathological state and core pathogenesis.[Results]Wendan Decoction originated from Banxia Shumi Decoction and was related to many classical prescriptions such as Banxia Shumi Decoction,ZHANG Zhongjing's"Xiaobanxia Decoction"and Shenshi's"Dajupi Decoction".The treatment object is the combined disease of gallbladder and stomach,which is caused by the weakness of Zangfu organs after a serious disease,and the core pathogenesis is"deficiency of cold in the middle-Jiao and the inversion of Jue Qi",and the main symptom is emesis.The prescription principle of Wendan Decoction emphasizes"warming the middle and reducing inversion,regulating Qi and relieving vexation,harmonizing Ying and Wei".The medication characteristics reflect"the combination of mild reduction and warming and clearing".The application indications include nausea,dizziness,chest fullness,irritability and other manifestations of syncope and Jue Qi inversion.[Conclusion]Wendan Decoction has multiple sources,and has good clinical effect in the treatment of"insomnia"disease with the core pathogenesis of combined disease of gallbladder and stomach.The medication has the characteristics of warming and descending,which is worth further investigation for the treatment of syndromes marked by Jue Qi inversion as the core manifestation.

Objective:To explore the clinical features, histopathological morphology, and differential diagnosis of lymphoepithelioma-like carcinoma with abnormal expression of follicular dendritic cell markers.Methods:From 2020 to 2021, 4 cases of lymphoepithelioma-like carcinoma with abnormal expression of follicular dendritic cell markers diagnosed in Fujian Cancer Hospital (2 cases) and the Second Affiliated Hospital of Fujian Medical University (2 cases) were collected. Different ancillary procedures such as HE, special stains, immunohistochemistry, and in situ hybridization techniques were used to assess the histopathological features and immunophenotypes. The clinical data were collected and literature was reviewed.Results:All 4 cases of lymphoepithelioma-like carcinoma with abnormal expression of follicular dendritic cell markers were male. They were 32, 45, 67 and 39 years old, respectively. The main clinical manifestations were bloody phlegm, abdominal pain, fatigue and anorexia. The clinical stages at diagnosis were stage Ⅳ (3 cases) and stage Ⅱ (1 case). Cases 2 and 3 had two pathological examinations at different sites, with a total of six pathological examinations. The histomorphology showed singly scattered or nests of tumor cells in a background of abundant small lymphocytes. The tumor cells were enlarged and pleomorphic, some appeared polygonal with inconspicuous cell borders, and they were arranged in a syncytial pattern. There were megakaryocytes, multinucleated tumor cells, and a few spindle-shaped cells seen. Atypical mitosis was commonly noted. By immunohistochemistry, the tumor cells were positive for CKpan(5/6), CK8/18(4/4), CAM5.2(2/5), CK-H(0/4), CK-L(3/4), EMA(4/5), CK5/6(3/6), p63(1/6), p40(1/6), E-cadherin (4/6), SSTR2(6/6), PD-L1(5/5), LCA(0/6), vimentin(5/6), CD2 (6/6), CD23(6/6), CD35(5/6), CXCL-13(4/5) and D2-40(1/5). The Ki-67 proliferative index was 60%-95%. In situ hybridization for EBER were all positive (6/6). Special stain for reticulin showed positive staining surrounding nests of tumor cells.Conclusions:The expression of follicular dendritic cell markers in lymphoepithelioma-like carcinoma is very rare, which may be related to EBV infection. Occasionally, it can overlap with follicular dendritic cell sarcoma by morphology and immunophenotype, which can lead to misdiagnosis. Only by combining clinical information, morphological characteristics and immunophenotype can an appropriate diagnosis be made.