Murine MHC-Deficient Nonobese Diabetic Mice Carrying Human HLA-DQ8 Develop Severe Myocarditis and Myositis in Response to Anti-PD-1 Immune Checkpoint Inhibitor Cancer Therapy.

Myocarditis has emerged as an immune-related adverse event of immune checkpoint inhibitor (ICI) cancer therapy associated with significant mortality. To ensure patients continue to safely benefit from life-saving cancer therapy, an understanding of fundamental immunological phenomena underlying ICI myocarditis is essential. We recently developed the NOD-cMHCI/II-/-.DQ8 mouse model that spontaneously develops myocarditis with lower mortality than observed in previous HLA-DQ8 NOD mouse strains. Our strain was rendered murine MHC class I and II deficient using CRISPR/Cas9 technology, making it a genetically clean platform for dissecting CD4+ T cell-mediated myocarditis in the absence of classically selected CD8+ T cells. These mice are highly susceptible to myocarditis and acute heart failure following anti-PD-1 ICI-induced treatment. Additionally, anti-PD-1 administration accelerates skeletal muscle myositis. Using histology, flow cytometry, adoptive transfers, and RNA sequencing analyses, we performed a thorough characterization of cardiac and skeletal muscle T cells, identifying shared and unique characteristics of both populations. Taken together, this report details a mouse model with features of a rare, but highly lethal clinical presentation of overlapping myocarditis and myositis following ICI therapy. This study sheds light on underlying immunological mechanisms in ICI myocarditis and provides the basis for further detailed analyses of diagnostic and therapeutic strategies.

Inhibition of RhoGEF/RhoA alleviates regorafenib resistance and cancer stemness via Hippo signaling pathway in hepatocellular carcinoma.

Patients with hepatocellular carcinoma (HCC) are vulnerable to drug resistance. Although drug resistance has been taken much attention to HCC therapy, little is known of regorafenib and regorafenib resistance (RR). This study aimed to determine the drug resistance pattern and the role of RhoA in RR. Two regorafenib-resistant cell lines were constructed based on Huh7 and Hep3B cell lines. In vitro and in vivo assays were conducted to study RhoA expression, the activity of Hippo signaling pathway and cancer stem cell (CSC) traits. The data showed that RhoA was highly expressed, Hippo signaling was hypoactivated and CSC traits were more prominent in RR cells. Inhibiting RhoA could reverse RR, and the alliance of RhoA inhibition and regorafenib synergistically attenuated CSC phenotype. Furthermore, inhibiting LARG/RhoA increased Kibra/NF2 complex formation, prevented YAP from shuttling into the nucleus and repressed CD44 mRNA expression. Clinically, the high expression of RhoA correlated with poor prognosis. LARG, RhoA, YAP1 and CD44 show positive correlation with each other. Thus, inhibition of RhoGEF/RhoA has the potential to reverse RR and repress CSC phenotype in HCC.

Predictive value of NoSAS questionnaire combined with the modified Mallampati grade for hypoxemia during routine sedation for gastrointestinal endoscopy.

BACKGROUND: The incidence of hypoxemia during painless gastrointestinal endoscopy remains a matter of concem. To date, there is no recognized simple method to predict hypoxemia in digestive endoscopic anesthesia. The NoSAS (neck circumference, obesity, snoring, age, sex) questionnaire, an objective and simple assessment scale used to assess obstructive sleep apnea (OSA), combined with the modified Mallampati grade (MMP), may have certain screening value. This combination may allow anesthesiologists to anticipate, manage, and consequently decrease the occurrence of hypoxemia. METHODS: This study was a prospective observational trial. The primary endpoint was the incidence of hypoxaemia defined as pulse oxygen saturation (SpO2) < 95% for 10 s. A total of 2207 patients admitted to our hospital for painless gastrointestinal endoscopy were studied. All patients were measured for age, height, weight, body mass index, neck circumference, snoring, MMP, and other parameters. Patients were divided into hypoxemic and non-hypoxemic groups based on the SpO2. The ROC curve was plotted to evaluate the screening value of the NoSAS questionnaire separately and combined with MMP for hypoxemia. The total NoSAS score was evaluated at cut-off points of 8 and 9. RESULTS: With a NoSAS score ≥ 8 as the critical value for analysis, the sensitivity for hypoxemia was 58.3%, the specificity was 88.4%, and the area under the ROC was 0.734 (P < 0.001, 95% CI: 0.708-0.759). With a NoSAS score ≥ 9 as a critical value, the sensitivity for hypoxemia was 36.50%, the specificity rose to 96.16%, and the area under the ROC was 0.663 (P < 0.001, 95% CI: 0.639-0.688). With the NoSAS Score combined with MMP for analysis, the sensitivity was 78.4%, the specificity was 84%, and the area under the ROC was 0.859 (P < 0.001, 95%CI:0.834-0.883). CONCLUSIONS: As a new screening tool, the NoSAS questionnaire is simple, convenient, and useful for screening hypoxemia. This questionnaire, when paired withMMP, is likely to be helpful for the screening of hypoxemia.

Propofol Suppresses Glioma Tumorigenesis by Regulating circ_0047688/miR-516b-5p/IFI30 Axis.

Propofol has recently attracted increasing attention for its anti-tumor property in cancers, including glioma. Circular RNAs (circRNAs) can act as key regulators in various cancers. However, the relationship between propofol and circ_0047688 in glioma is still unclear. Cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), and colony formation assays. Cell migration and invasion were determined using transwell assay. Cell apoptosis was detected by flow cytometry. Protein levels and RNA levels were detected by western blot assay and real-time quantitative polymerase chain reaction (RT­qPCR), respectively. The intermolecular interaction was predicted by bioinformatics analysis and verified by dual-luciferase reporter assay. A mouse xenograft model was established for in vivo experiments. Propofol inhibited cell proliferation, migration, and invasion and accelerated apoptosis in glioma cells. Circ_0047688 was upregulated in glioma tissues and cells, and propofol downregulated circ_0047688 in a dose-dependent manner. Circ_0047688 knockdown inhibited glioma cell progression and its overexpression abated the anti-tumor role of propofol in glioma cells. Moreover, miR-516b-5p was a direct target of circ_0047688, and circ_0047688 promoted glioma cell progression by sponging miR-516b-5p. In addition, IFI30 was a direct target of miR-516b-5p, and miR-516b-5p inhibited glioma cell malignant behaviors by targeting IFI30 in propofol-treated cells. Furthermore, circ_0047688 overexpression could weaken the anti-tumor role of propofol in vivo. Propofol inhibited glioma progression via modulating circ_0047688/miR-516b-5p/IFI30 axis, providing a potential therapeutic strategy for treatment of glioma.

Structure, dynamics and transport behavior of migrating corrosion inhibitors on the surface of calcium silicate hydrate: a molecular dynamics study.

The incorporation of a corrosion inhibitor into a cement-based material can enhance the durability of the reinforced concrete. In this study, molecular dynamics simulation is utilized to study the interfacial structure and dynamic behavior of a solution with three migrating corrosion inhibitors (MCI) functionalized by hydroxyl (-OH), carboxyl (-COO-), and phenyl (-PH) groups in calcium silicate hydrate (CSH) gel pores. The transport rate of inhibitors is greatly dependent on the polarity of the functional group: -PH > -OH > -COO-. The slow migration rate of the inhibitor with -OH and -COO- is attributed to the chemical bond formed between CSH and MCI. The silicate chains near the CSH surface can provide plenty of non-bridging oxygen sites to accept the H-bond from the hydroxyl group in the inhibitor molecule. The surface calcium atom can capture the -COO- by forming an ionic COO-Ca bond. Furthermore, the hydration structure of the inhibitor molecule also influences its transport properties. The inhibitor functionalized by the carboxyl group, associating with the neighboring water molecules, forms ion-water clusters, and the inhibitor molecule and its hydration shell with a long resident time retard the migration rate. Hopefully, this study is able to provide molecules for the development of a migration-type corrosion inhibitor to elongate the service life of cement-based materials.

Hinokiflavone induces apoptosis via activating mitochondrial ROS/JNK/caspase pathway and inhibiting NF-κB activity in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the sixth most common malignancy with limited treatment options. Hinokiflavone (HF), a natural biflavonoid, has shown to inhibit the proliferation of melanoma, whereas its antitumour effect against HCC and the underlying mechanisms remain elusive. Here, we aimed at evaluating its antitumour effect against HCC in both in vitro and in vivo. Cell counting kit 8, colony formation assay, PI/RNase staining and Western blotting revealed that HF inhibited the proliferation of HCC cells via G0/G1 cell cycle arrest with p21/p53 up-regulation. DAPI staining, Annexin V-FITC/PI staining and Western blotting confirmed that HF triggered caspase-dependent apoptosis. Moreover, HF increased the levels of mitochondrial reactive oxygen species (mtROS) and activated c-Jun N-terminal kinase (JNK) pathway, as measured by MitoSOX Red staining and Western blotting. After respectively inhibiting mtROS (Mito-TEMPO) and JNK (SP600125), HF-induced apoptosis was reversed. Additionally, Western blotting documented that HF suppressed nuclear factor kappa B (NF-κB) activity and the anti-apoptotic genes downstream, contributing to cell apoptosis. Finally, in vivo studies demonstrated that HF significantly impaired tumour growth in HCC xenograft. Collectively, these findings suggested that HF induced apoptosis through activating mtROS/JNK/caspase pathway and inhibiting NF-κB signalling, which may represent a novel therapeutic agent for treating HCC.

Hypoxic induction of vasculogenic mimicry in hepatocellular carcinoma: role of HIF-1 α, RhoA/ROCK and Rac1/PAK signaling.

BACKGROUND: Vasculogenic mimicry (VM), defined as a capability of aggressive tumor Cells to mimic embryonic vasculogenic networks, caused poor prognosis in hepatocellular carcinoma (HCC). Rho kinases (ROCK), p21-activated kinase (PAK), hypoxia or epithelial-mesenchymal transition (EMT) contributed to the VM potential. However, the details underlying these biological behaviors have not been completely elucidated. METHODS: Kaplan-Meier analysis was conducted to predict relationship with hypoxia Inducible factor (HIF-1α), EMT related markers: Vimentin and patient prognosis. CD34/periodic acid-Schiff (PAS) double staining was examined to differentiate VM-positive (VM+) and VM-negative (VM-) samples. Cells were cultured under controlled hypoxic environments (1% O2) or normoxic conditions. The effect of hypoxia on RhoA/ROCK, Rac1/PAK and EMT were evaluated by real time-qPCR and western blot. HIF-1α small interfering RNA (siRNA), overexpressed or short hairpin RNA (shRNA) of ROCK and kinase inhibitors were used to explore the effect of HIF-1α, RhoA/ROCK, Rac1/PAK and Vimentin on VM. RESULTS: HIF-1α or Vimentin was upregulated in VM+ HCC tissues, compared to non-cancerous tissues (P < 0.01), and patients with high expression of HIF-1α or Vimentin had worse prognosis (P < 0.001). We showed hypoxia induced RhoA/ROCK and Rac1/PAK signaling transduction, and EMT could be repressed by HIF-1α siRNA. Notably, RhoA/ROCK or Rac1/PAK stabilized HIF-1α in hypoxia, whereas HIF-1α did not significantly altered RhoA/ROCK or Rac1/PAK signaling in hypoxia. Moreover, we found distinct roles of ROCK1, ROCK2 and PAK in regulating Vimentin phosphorylation. CONCLUSIONS: RhoA/ROCK and Rac/PAK signaling played crucial roles in hypoxia-induced VM via Ser72 and Ser56 Vimentin phosphorylation in HCC.

Rho kinase mediates transforming growth factor-ß1-induced vasculogenic mimicry formation: involvement of the epithelial-mesenchymal transition and cancer stemness activity.

Vasculogenic mimicry (VM), a newly defined pattern of tumor blood supply, has been identified in several malignant tumors, including hepatocellular carcinoma (HCC). Rho kinase (ROCK) plays an important role in various types of cancers. However, whether ROCK participates in transforming growth factor-ß1 (TGF-ß1)-induced VM formation is unclear. Here, we evaluated the role of ROCK in TGF-ß1-induced VM formation in HCC. Our findings showed that the TGF-ß1/ROCK signaling pathway is involved in VM formation by inducing the epithelial-mesenchymal transition. Furthermore, TGF-ß1 and ROCK were found to play distinct roles in the cancer stem cell phenotype during VM formation. These results provide insights into potential antitumor therapies for inhibiting VM by targeting the TGF-ß1/ROCK signaling pathway in HCC.

Overlapping and unique roles played by ROCK1 and 2 in the modulation of coding and long noncoding RNA expression.

BACKGROUND: Our previous study described the crucial role of Rho-associated coiled-coil containing-kinases (ROCK) in hepatocellular carcinoma (HCC). However, the potential significance of long noncoding RNA downstream of ROCK is largely unknown. Here, a comprehensive comparative bioinformatics analysis of a microarray of an MHCC-97H cell line overexpressing ROCK1 or ROCK2 was performed. RESULTS: Numerous lncRNAs and mRNAs were deregulated by Rho-associated coiled-coil containing kinases 1 and 2. These results were consistent with the qRT-PCR results. Compared with MHCC-97H-Con, which was transfected with a null vector, the GO analysis revealed differentially expressed mRNAs (DEmRNAs) in MHCC-97H-ROCK1 (ROCK1 was overexpressed) enriched in apoptotic cell clearance, the cyclooxygenase pathway and bone trabecula morphogenesis; the DEmRNAs in MHCC-97H-ROCK2 (ROCK2 was overexpressed) were enriched in VEGF production, chemokine-associated signaling pathways, acute inflammatory response and vasoconstriction. Compared with MHCC-97H-ROCK2, the DEmRNAs in MHCC-97H-ROCK1 were involved in the JAK-STAT cascade, the Akt signaling pathway and the activity of several different peptidases. The pathway analysis of ROCK1 and ROCK2 revealed an overlap in the VEGF signaling pathway, ECM-receptor interaction, and adhesion and differences in the PPAR signaling pathway and mismatch repair. The predicted targets of the differentially expressed lncRNA (DElncRNAs) were enriched in the p53 signaling pathway, Jak-STAT signaling pathway, etc. Several hub DElncRNAs were identified. CONCLUSIONS: ROCK1 and 2 modulate the expression of numerous mRNAs and lncRNAs and may participate in several signaling pathways in HCC. Several hub molecules were identified in the lncRNA-mRNA networks. Our results provide baseline data for ROCK1 and 2 regulation in HCC that might have implications for further research.

Low-, high-coverage, and two-stage DNA sequencing in the design of the genetic association study.

Next-generation sequencing-based genetic association study (GAS) is a powerful tool to identify candidate disease variants and genomic regions. Although low-coverage sequencing offers low cost but inadequacy in calling rare variants, high coverage is able to detect essentially every variant but at a high cost. Two-stage sequencing may be an economical way to conduct GAS without losing power. In two-stage sequencing, an affordable number of samples are sequenced at high coverage as the reference panel, then to impute in a larger sample is sequenced at low coverage. As unit sequencing costs continue to decrease, investigators can now conduct GAS with more flexible sequencing depths. Here, we systematically evaluate the effect of the read depth and sample size on the variant discovery power and association power for study designs using low-coverage, high-coverage, and two-stage sequencing. We consider 12 low-coverage, 12 high-coverage, and 51 two-stage design scenarios with the read depth varying from 0.5× to 80×. With state-of-the-art simulation and analysis packages and in-house scripts, we simulate the complete study process from DNA sequencing to SNP (single nucleotide polymorphism) calling and association testing. Our results show that with appropriate allocation of sequencing effort, two-stage sequencing is an effective approach for conducting GAS. We provide practical guidelines for investigators to plan the optimum sequencing-based GAS including two-stage sequencing design given their specific constraints of sequencing investment.

A Rho-kinase inhibitor reverses learning and memory deficits in a Rat model of chronic cerebral ischemia by altering Bcl-2/Bax-NMDAR signaling in the cerebral cortex.

The current study investigated whether a Rho-kinase inhibitor alleviated impairments in a rat model of chronic cerebral ischemia and examined the specific pathological mechanisms by which Rho-kinase impacts neuronal damage and cognitive dysfunction. Adult Sprague-Dawley rats underwent permanent bilateral carotid artery occlusion (BCAO) to establish our chronic cerebral ischemia model. Chronic Y27632 administration reversed the abnormal behaviors of BCAO-treated rats in the Morris water maze. We performed Western blot analyses of the apoptosis-related proteins Bcl-2 and Bax to examine the potential mechanism underlying the beneficial effects of Y27632 on cerebral ischemia and showed for the first time that Y27632 reversed the decrease in the Bcl-2/Bax ratio in BCAO model rats. Y27632 restored the depression of NR2A- and NR2B-containing N-methyl-d-aspartate receptors (NMDARs) in the cerebral cortex of BCAO model rats. We also investigated these effects on middle cerebral artery occlusion (MCAO) model rats and observed some differences between the two models. In summary, our data provide evidence supporting the hypothesis that Rho-kinase inhibitors exert neuroprotective effects on cerebral ischemia. The Bcl-2/Bax-NMDAR signaling pathway in the cerebral cortex may be responsible for the protective effects of the Rho-kinase inhibitor, and this pathway may represent a pharmacological target for curative clinical strategies.

Comparison of statistical methods for subnetwork detection in the integration of gene expression and protein interaction network.

BACKGROUND: With the advancement of high-throughput technologies and enrichment of popular public databases, more and more research focuses of bioinformatics research have been on computational integration of network and gene expression profiles for extracting context-dependent active subnetworks. Many methods for subnetwork searching have been developed. Scoring and searching algorithms present a range of computational considerations and implementations. The primary goal of present study is to comprehensively evaluate the performance of different subnetwork detection methods. Eleven popular methods were selected for comprehensive comparison. RESULTS: First, taking into account the dependence of genes given a protein-protein interaction (PPI) network, we simulated microarray gene expression data under case and control conditions. Then each method was applied to the simulated data for subnetwork identification. Second, a large microarray data set of prostate cancer was used to assess the practical performance of each method. Using both simulation studies and a real data application, we evaluated the performance of different methods in terms of recall and precision. CONCLUSIONS: jActiveModules, PinnacleZ and WMAXC performed well in identifying subnetwork with relative high precision and recall. BioNet performed very well only in precision. As none of methods outperformed other methods overall, users should choose an appropriate method based on the purposes of their studies.

Effects of 630 nm Red and 460 nm Blue Light Emitting Diode Irradiation on Healing of the Skin Wound in Japanese Big-ear White Rabbit.

Objective To observe the effects of 630 nm red light and 460 nm blue light emitting diode irradiation on the healing of skin wounds in Japanese big-ear white rabbits. Methods The skin wound model was established with 8 Japanese big-ear white rabbits. Three parts of vulnus in each rabbit were used:two parts of vulnus were irradiated vertically by red and blue LED light,respectively(15 min/time),and the distance between lights and wounds was 15 cm;the 3rd part of the wound was used as a control. On the 21st day of the wounds exposure to light,the number of healing wounds and the percentage of healing area were recorded and the treatment effect of these two light sources was compared. HE staining was used to analyze the newborn tissue structure. Masson staining was used to observe the proliferation of skin collagen fibers. Immuohistochemical staining was used to analyze fibroblast growth factor(FGF),epidermal growth factor(EGF),endothelial growth factor(CD31),proliferating cell nuclear antigen(Ki-67),and inflammatory cytokines(CD68)infiltration in the skin. Results The healing rate in the red light,blue light,and control groups was 50.0%(4/8),25.0%(2/8),and 12.5%(1/8),respectively. Since the 12th day after modeling,the healing area percentage in the red light group was significantly higher than those in the blue light and control groups(P<0.05,P<0.01). On the 21st day after modeling,the skin thickness of the red light group was(2.95±0.34)mm,which was significantly higher than that in control group [(2.52±0.42)mm;F=3.182,P=0.016)]. The average optical density of collagen fibers was 0.15±0.03 in red light group,which was significantly higher than that of the blue light group(0.09±0.01;F=7.316,P=0.012)and control(0.07±0.01;F=7.316,P=0.003). The results of immunohistochemistry showed the expression levels of EGF,FGF,CD31 antigen,and Ki-67 in the red light group were significantly higher than those in the blue light and control groups,whereas the CD68 expression was significantly lower(P<0.05 or P<0.01). Conclusion LED red light irradiation can promote the healing of skin wounds in Japanese big-ear white rabbits,which may be achieved by the effect of red light irradiation in stimulating the proliferation of skin epidermal cells,vascular endothelial cells,and fiberous tissue.

Network-based proteomic analysis for postmenopausal osteoporosis in Caucasian females.

Menopause is one of the crucial physiological events during the life of a woman. Transition of menopause status is accompanied by increased risks of various health problems such as osteoporosis. Peripheral blood monocytes can differentiate into osteoclasts and produce cytokines important for osteoclast activity. With quantitative proteomics LC-nano-ESI-MS(E) (where MS(E) is elevated-energy MS), we performed protein expression profiling of peripheral blood monocytes in 42 postmenopausal women with discordant bone mineral density (BMD) levels. Traditional comparative analysis showed proteins encoded by four genes (LOC654188, PPIA, TAGLN2, YWHAB) and three genes (LMNB1, ANXA2P2, ANXA2) were significantly down- and upregulated, respectively, in extremely low- versus high-BMD subjects. To study functionally orchestrating groups of detected proteins in the form of networks, we performed weighted gene coexpression network analysis and gene set enrichment analysis. Weighted gene coexpression network analysis showed that the module including the annexin gene family was most significantly correlated with low BMD, and the lipid-binding related GO terms were enriched in this identified module. Gene set enrichment analysis revealed that two significantly enriched gene sets may be involved in postmenopausal BMD variation by regulating pro-inflammatory cytokines activities. To gain more insights into the proteomics data generated, we performed integrative analyses of the datasets available to us at the genome (DNA level), transcriptome (RNA level), and proteome levels jointly.

An integrative imputation method based on multi-omics datasets.

BACKGROUND: Integrative analysis of multi-omics data is becoming increasingly important to unravel functional mechanisms of complex diseases. However, the currently available multi-omics datasets inevitably suffer from missing values due to technical limitations and various constrains in experiments. These missing values severely hinder integrative analysis of multi-omics data. Current imputation methods mainly focus on using single omics data while ignoring biological interconnections and information imbedded in multi-omics data sets. RESULTS: In this study, a novel multi-omics imputation method was proposed to integrate multiple correlated omics datasets for improving the imputation accuracy. Our method was designed to: 1) combine the estimates of missing value from individual omics data itself as well as from other omics, and 2) simultaneously impute multiple missing omics datasets by an iterative algorithm. We compared our method with five imputation methods using single omics data at different noise levels, sample sizes and data missing rates. The results demonstrated the advantage and efficiency of our method, consistently in terms of the imputation error and the recovery of mRNA-miRNA network structure. CONCLUSIONS: We concluded that our proposed imputation method can utilize more biological information to minimize the imputation error and thus can improve the performance of downstream analysis such as genetic regulatory network construction.

Multistage genome-wide association meta-analyses identified two new loci for bone mineral density.

Aiming to identify novel genetic variants and to confirm previously identified genetic variants associated with bone mineral density (BMD), we conducted a three-stage genome-wide association (GWA) meta-analysis in 27 061 study subjects. Stage 1 meta-analyzed seven GWA samples and 11 140 subjects for BMDs at the lumbar spine, hip and femoral neck, followed by a Stage 2 in silico replication of 33 SNPs in 9258 subjects, and by a Stage 3 de novo validation of three SNPs in 6663 subjects. Combining evidence from all the stages, we have identified two novel loci that have not been reported previously at the genome-wide significance (GWS; 5.0 × 10(-8)) level: 14q24.2 (rs227425, P-value 3.98 × 10(-13), SMOC1) in the combined sample of males and females and 21q22.13 (rs170183, P-value 4.15 × 10(-9), CLDN14) in the female-specific sample. The two newly identified SNPs were also significant in the GEnetic Factors for OSteoporosis consortium (GEFOS, n = 32 960) summary results. We have also independently confirmed 13 previously reported loci at the GWS level: 1p36.12 (ZBTB40), 1p31.3 (GPR177), 4p16.3 (FGFRL1), 4q22.1 (MEPE), 5q14.3 (MEF2C), 6q25.1 (C6orf97, ESR1), 7q21.3 (FLJ42280, SHFM1), 7q31.31 (FAM3C, WNT16), 8q24.12 (TNFRSF11B), 11p15.3 (SOX6), 11q13.4 (LRP5), 13q14.11 (AKAP11) and 16q24 (FOXL1). Gene expression analysis in osteogenic cells implied potential functional association of the two candidate genes (SMOC1 and CLDN14) in bone metabolism. Our findings independently confirm previously identified biological pathways underlying bone metabolism and contribute to the discovery of novel pathways, thus providing valuable insights into the intervention and treatment of osteoporosis.

Positive selection of B10 cells is determined by BCR specificity and signaling strength.

B10 cells, a regulatory B cell subset, negatively regulate immune responses in an IL-10-dependent manner. However, the mechanism of B10 cell development is unclear. We found that B10 cells mainly identified self-antigens. TgVH3B4 transgenic mice, whose VH was derived from an actin-reactive natural antibody, exhibit elevated numbers of actin-binding B10 cells. Immunization of TgVH3B4 mice with actin induced elevated B10 cell numbers in an antigen-specific manner, indicating positive selection of B10 cells by self-antigens. Furthermore, higher BCR signaling strength facilitated B10 cell development. We also observed that actin-reactive IgG levels were unchanged in TgVH3B4 mice after immunization with actin in contrast to the elevated OVA-reactive IgG level after immunization with OVA, indicating that B10 cells acted in an antigen-specific manner to inhibit the immune response. Our data demonstrate for the first time that B10 cells are positively selected by self-reactivity and that higher BCR signaling strength promotes B10 cell development.

Incarvine C suppresses proliferation and vasculogenic mimicry of hepatocellular carcinoma cells via targeting ROCK inhibition.

BACKGROUND: Studies have described vasculogenic mimicry (VM) as an alternative circulatory system to blood vessels in multiple malignant tumor types, including hepatocellular carcinoma (HCC). In the current study, we aimed to seek novel and more efficient treatment strategies by targeting VM and explore the underlying mechanisms in HCC cells. METHODS: Cell counting kit-8 (CCK-8) assay and colony survival assay were performed to explore the inhibitory effect of incarvine C (IVC) on human cancer cell proliferation. Flow cytometry was performed to analyze the cell cycle distribution after DNA staining and cell apoptosis by the Annexin V-PE and 7-AAD assay. The effect of IVC on Rho-associated, coiled-coil-containing protein kinase (ROCK) was determined by western blotting and stress fiber formation assay. The inhibitory role of IVC on MHCC97H cell VM formation was determined by formation of tubular network structures on Matrigel in vitro, real time-qPCR, confocal microscopy and western blotting techniques. RESULTS: We explored an anti-metastatic HCC agent, IVC, derived from traditional Chinese medicinal herbs, and found that IVC dose-dependently inhibited the growth of MHCC97H cells. IVC induced MHCC97H cell cycle arrest at G1 transition, which was associated with cyclin-dependent kinase 2 (CDK-2)/cyclin-E1 degradation and p21/p53 up-regulation. In addition, IVC induced apoptotic death of MHCC97H cells. Furthermore, IVC strongly suppressed the phosphorylation of the ROCK substrate myosin phosphatase target subunit-1 (MYPT-1) and ROCK-mediated actin fiber formation. Finally, IVC inhibited cell-dominant tube formation in vitro, which was accompanied with the down-regulation of VM-key factors as detected by real time-qPCR and immunofluorescence. CONCLUSIONS: Taken together, the effective inhibitory effect of IVC on MHCC97H cell proliferation and neovascularization was associated with ROCK inhibition, suggesting that IVC may be a new potential drug candidate for the treatment of HCC.

Jacarelhyperol A induced apoptosis in leukaemia cancer cell through inhibition the activity of Bcl-2 proteins.

BACKGROUND: Hypericum japonicum Thunb. ex Murray is widely used as an herbal medicine for the treatment of hepatitis and tumours in China. However, the molecular mechanisms of its effects are unclear. Our previous research showed that extracts of H. japonicum can induce apoptosis in leukaemia cells. We also previously systematically analysed and isolated the chemical composition of H. japonicum. METHODS: The fluorescence polarisation experiment was used to screen for inhibitors of Bcl-2 proteins which are proved as key proteins in apoptosis. The binding mode was modelled by molecular docking. We investigated the proliferation attenuating and apoptosis inducing effects of active compound on cancer cells by MTT assay and flow cytometry analysis. Activation of caspases were tested by Western blot. A broad-spectrum caspase inhibitor Z-VAD-FMK was used to investigate the caspases-dependence. In addition, co-immunoprecipitation was performed to analyse the inhibition of heterodimerization between anti-apoptotic Bcl-2 proteins with pro-apoptotic proteins. Moreover, in vivo activity was tested in a mouse xenograph tumour model. RESULT: Jacarelhyperol A (Jac-A), a characteristic constituent of H. japonicum, was identified as a potential Bcl-2 inhibitor. Jac-A showed binding affinities to Bcl-xL, Bcl-2, and Mcl-1 with Ki values of 0.46 µM, 0.43 µM, and 1.69 µM, respectively. This is consistent with computational modelling results, which show that Jac-A presents a favorable binding mode with Bcl-xL in the BH3-binding pocket. In addition, Jac-A showed potential growth inhibitory activity in leukaemia cells with IC50 values from 1.52 to 6.92 µM and significantly induced apoptosis of K562 cells by promoting release of cytochrome c and activating the caspases. Jac-A also been proved that its effect is partly caspases-dependent and can disrupt the heterodimerization between anti-apoptotic Bcl-2 proteins with pro-apoptotic proteins. Moreover, Jac-A dose-dependently inhibited human K562 cell growth in a mouse xenograph tumour model with low toxicity. CONCLUSION: In this study, a characteristic constituent of H. japonicum, Jac-A, was shown to induce apoptosis in leukaemia cells by mediating the Bcl-2 proteins. Therefore, we propose a new lead compound for cancer therapy with a low toxicity, and have provided evidence for using H. japonicum as an anti-cancer herb.