High expression levels of SLFN11 correlate with the sensitivity of human cancer cells to DNA-damaging agents. However, little is known about the underlying mechanism. Here, we show that SLFN11 interacts directly with RPA1 and is recruited to sites of DNA damage in an RPA1-dependent manner. Furthermore, we establish that SLFN11 inhibits checkpoint maintenance and homologous recombination repair by promoting the destabilization of the RPA-ssDNA complex, thereby sensitizing cancer cell lines expressing high endogenous levels of SLFN11 to DNA-damaging agents. Finally, we demonstrate that the RPA1-binding ability of SLFN11 is required for its function in the DNA damage response. Our findings not only provide novel insight into the molecular mechanisms underlying the drug sensitivity of cancer cell lines expressing SLFN11 at high levels, but also suggest that SLFN11 expression can serve as a biomarker to predict responses to DNA-damaging therapeutic agents.

Osteoporosis is a disease characterized by low bone mass and progressive destruction of bone microstructure, resulting in increased the risk of fracture. Previous studies have demonstrated the effect of naringin (NG) or treadmill exercise (EX) on osteoporosis, however, reports about effects of NG plus EX on osteoporosis are limited. This study was designed to investigate the impact of combined treatment with naringin and treadmill exercise on osteoporosis in ovariectomized (OVX) rats. Three months after bilateral ovariectomy, Seventy-five rats were randomly assigned to the following treatment groups: OVX, sham-operated (SHAM), NG, EX, or NG plus EX treatment. Treatments were administered for 60 days. Bone metabolism, bone mineral density, trabecular bone parameters, immunohistochemistry, and the bone strength were evaluated. Compared to the OVX groups, all treatments increased bone volume (BV/TV), trabecula number (Tb.N), trabecula thickness (Tb.Th), bone mineral density (BMD), and mechanical strength. NG + EX showed the strongest effects on BV/TV, Tb.Th, and biomechanical strength. Additionally, decreased C-terminal telopeptides of type I collagen (CTX-1) and enhanced osteocalcin (OCN) expression were observed in the NG + EX group. The present study demonstrates that the NG + EX may have a therapeutic advantage over each monotherapy for the treatment of osteoporosis.

Organ size determination is one of the most intriguing unsolved mysteries in biology. Aberrant activation of the major effector and transcription co-activator YAP in the Hippo pathway causes drastic organ enlargement in development and underlies tumorigenesis in many human cancers. However, how robust YAP activation is achieved during organ size control remains elusive. Here we report that the YAP signaling is sustained through a novel microRNA-dependent positive feedback loop. miR-130a, which is directly induced by YAP, could effectively repress VGLL4, an inhibitor of YAP activity, thereby amplifying the YAP signals. Inhibition of miR-130a reversed liver size enlargement induced by Hippo pathway inactivation and blocked YAP-induced tumorigenesis. Furthermore, the Drosophila Hippo pathway target bantam functionally mimics miR-130a by repressing the VGLL4 homolog SdBP/Tgi. These findings reveal an evolutionarily conserved positive feedback mechanism underlying robustness of the Hippo pathway in size control and tumorigenesis.

BMP signaling mediated by ACVR1 plays a critical role for development of multiple structures including the cardiovascular and skeletal systems. While deficient ACVR1 signaling impairs normal embryonic development, hyperactive ACVR1 function (R206H in humans and Q207D mutation in mice, ca-ACVR1) results in formation of heterotopic ossification (HO). We developed a mouse line, which conditionally expresses ca-ACVR1 with Nfatc1-Cre(+) transgene. Mutant mice developed ectopic cartilage and bone at the distal joints of the extremities including the interphalangeal joints and hind limb ankles as early as P4 in the absence of trauma or exogenous bone morphogenetic protein (BMP) administration. Micro-CT showed that even at later time points (up to P40), cartilage and bone development persisted at the affected joints most prominently in the ankle. Interestingly, this phenotype was not present in areas of bone outside of the joints - tibia are normal in mutants and littermate controls away from the ankle. These findings demonstrate that this model may allow for further studies of heterotopic ossification, which does not require the use of stem cells, direct trauma or activation with exogenous Cre gene administration.

Many studies have demonstrated that leukoaraiosis is associated with impaired cerebrovascular reserve function. However, the definitive hemodynamic changes that occur in leukoaraiosis are not clear, and there are many controversies. This study aimed to investigate hemodynamic changes in symptomatic leukoaraiosis using transcranial Doppler ultrasonography and the breath-holding test in a Chinese Han population, from northern China. A total of 203 patients who were diagnosed with ischemic stroke or clinical chronic progressive ischemic symptoms were enrolled in this study, including 97 males and 106 females, with an age range of 43-93 years. The severity of leukoaraiosis was evaluated according to the Fazekas grading scale, and patients were divided into four groups accordingly. Grade 0 was no leukoaraiosis, and grades I, II, and III were mild, moderate, and severe leukoaraiosis, respectively, with 44, 79, 44, and 36 cases in each group. Transcranial Doppler ultrasonography and the breath-holding test were performed. The mean blood flow velocity of the bilateral middle cerebral artery was measured and the breath-holding index was calculated. The breath holding index was correlated with leukoaraiosis severity and cognitive impairment. Patients with a low breath holding index presented poor performance in the Montreal Cognitive Assessment (MoCA) and executive function tests. That is, the lower the breath holding index, the lower the scores for the MoCA and the higher for the trail-making test Parts A and B. These results indicate that the breath-holding index is a useful parameter for the evaluation of cerebrovascular reserve impairment in patients with leukoaraiosis. In addition, the breath-holding index can reflect cognitive dysfunction, providing a new insight into the pathophysiology of leukoaraiosis. This study was approved by the Ethics Committee of the Fifth People's Hospital of Shenyang, China (approval No. 20160301) and registered in the Chinese Clinical Trial Registry (registration number: ChiCTR1800014421).

Several methods have been developed to improve the efficacy of foot-and-mouth disease virus (FMDV) vaccine. The study aims to determine whether aqueous extracts of Artemisia rupestris L. (AEAR) as an immunoactivator in combination with inactivated FMDV vaccine can promote immune responses in mice. Intramuscular co-immunization in ICR mice with different doses of AEAR plus FMDV vaccine could substantially improve the FMDV-specific antibody production (IgG, IgG1, and IgG2a) and lead to significant lymphocyte proliferative responses. Th1-type immune responses were also observed, including proliferative responses of CD8+, CD4+, CD4+CD44+, and CD8+CD44+ T cells and the killing efficacy of cytotoxic T lymphocyte (CTL) responses. AEAR also elicited the higher levels of IL-4 and IFN-γ in CD4+ T cells as well as the higher level of IFN-γ in CD8+ T cells. The medium dose of AEAR induced the significant adjuvant activity. Further tests in mice indicated that AEAR could activate DCs maturation by increasing the expression levels of co-stimulatory molecules (CD40, CD86, CD80, and MHC-II) on dendritic cells (DCs) from splenocytes and reduce the activity of regulatory T cells (Treg). Abnormal behaviors, side effects or death were not observed in immunized mice. AEAR could boost humoral and cell-mediated immunity elicited by FMDV vaccine, especially Th1-type immune responses.

As a new tumor marker, the microRNA-21 (miRNA21) level can provide important information for early diagnosis, drug treatment, and prognosis of gastric cancer. With the tool of molecular beacons which can hybridize specifically with target miRNA-21 and generate fluorescence signal change, this paper develops a direct, simple, and rapid method for miRNA-21 detection with detection limit of 0.5 nM. Under the optimal conditions, the method was used to detect the expression of miRNA-21 in tumor cells and tissues. The results showed significant differences of miRNA-21 levels in tumor cells which have different origins and different degree of malignancy. In 8 cases of gastric cancer tissues and adjacent tissues, the level of miRNA-21 in 6 cases was higher than that in adjacent tissues, 1 case had lower expression level than that in adjacent tissues, and 1 case had no significant difference. Furthermore, qRT-PCR method was used to verify the detection results based on the fluorescent probe detection method. The consistent results show that the molecular beacon assay has a good prospect in direct and rapid detection of miRNA-21 expression and will be widely used in the functional research and clinical diagnosis of microRNA.

Rho GTPase activating protein 9 (ARHGAP9), a member of RhoGAP family, has been identified as a RhoGAP for Cdc42 and Rac1. Here, we aimed to clarify the expression and functional role of ARHGAP9 in hepatocellular carcinoma (HCC). By analyzing TCGA (The Cancer Genome Atlas) LIHC (liver hepatocellular carcinoma) database, we found that ARHGAP9 expression was lower in HCC tissues than in normal liver tissues, and that patients with ARHGAP9 lower expression had a significant shorter overall survival time than those with ARHGAP9 higher expression. Cell counting kit-8 (CCK-8), transwell assays and in vivo experimental lung metastasis assay revealed that ARHGAP9 overexpression could inhibit HCC cell proliferation, migration and invasion, as well as HCC lung metastases. By next-generation RNA-sequencing, we identified that a transcription factor, Forkhead Box J2 (FOXJ2), was significantly induced by ARHGAP9 overexpression in HepG2 cells. Ectopic expression of FOXJ2 in HCC cell lines also exerted inhibitory effects on cell migration and invasion. Moreover, the inhibitory effects of ARHGAP9 on HCC cell migration and invasion was significantly attenuated by FOXJ2 knockdown. Luciferase reporter assay demonstrated that ARHGAP9 enhanced the transcription of E-cadherin (CDH1) via FOXJ2. Chromatin immunoprecipitation (ChIP) assay demonstrated that FOXJ2 modulated the transcription of E-cadherin (CDH1) by directly binding to its promoter. Furthermore, Pearson's correlation analysis indicated that the mRNA levels of ARHGAP9 in HCC tissues were positively correlated with the mRNA levels of FOXJ2 and CDH1. These data clearly show that ARHGAP9/FOXJ2 inhibit cell migration and invasion during HCC development via inducing the transcription of CDH1.

The role of arsenic trioxide (As2O3) in inhibiting immune rejection and prolonging islet allograft survival has been identified in islet allotransplantation. This study aims to explore the role of As2O3 in islet xenotransplantation and the action mechanism. The streptozotocin (STZ) was used in C57BL/6 mice to induce the type 1 diabetes mellitus (T1DM) for xenotransplantation models establishment. Donor islets were isolated by digesting. The flow cytometry (FCM) was used to analyze lymphocyte types. The blood sugar level was detected by using intraperitoneal glucose tolerance test (IPGTT). The serum level of cytokines was determined by the enzyme-linked immunosorbent assay (ELIZA). The cell proliferation was measured by MTT assay. The mRNA levels were quantified with qRT-PCR. As2O3 prolonged the survival of the recipient mice but had no influence on body weight. As2O3 protected the function of xenograft in insulin secretion and suppressed immune rejection of recipient. As2O3 inhibited proliferation of T lymphocyte and increased the proportion of Foxp3+ regulatory T cells in recipient mice. As2O3 inhibited activation and promoted clonal anergy of T lymphocyte. As2O3 decreased total number of B cells and reduced partial antibody levels in recipient mice. As2O3 and leflunomide showed a synergistic effect in suppressing islet xenotransplant rejection. As2O3 prolongs islet xenograft survival by inhibiting cellular immune response, and increasing Foxp3+ regulatory T cells, while decreasing partial antibody levels in serum.

Focal segmental glomerulosclerosis (FSGS) comprises a group of uncommon disorders that present with marked proteinuria, nephrotic syndrome, progressive renal failure and characteristic glomerular lesions on histopathology. The current standard of care for patients with FSGS include immunosuppressive drugs such as glucocorticoids followed by calcineurin inhibitors, if needed for intolerance or inadequate response to glucocorticoids. Renin-angiotensin-aldosterone (RAAS) blockers are also used to control proteinuria, an important signature of FSGS. Existing treatments, however, achieved only limited success. Despite best care, treatment failure is common and FSGS is causal in a significant proportion of end stage renal disease. Thus, an unmet need exists for novel disease modifying treatments for FSGS. We employed two widely-used murine models of FSGS to test the hypothesis that systemic inhibition of chemokine receptor CCR2 would have therapeutic benefit. Here we report that administration CCX872, a potent and selective small molecule antagonist of CCR2, achieved rapid and sustained attenuation of renal damage as determined by urine albumin excretion and improved histopathological outcome. Therapeutic benefit was present when CCX872 was used as a single therapy, and moreover, the combination of CCX872 and RAAS blockade was statistically more effective than RAAS blockade alone. In addition, the combination of CCR2 and RAAS blockade was equally as effective as endothelin receptor inhibition. We conclude that specific inhibition of CCR2 is effective in the Adriamycin-induced and 5/6 nephrectomy murine models of FSGS, and thus holds promise as a mechanistically distinct therapeutic addition to the treatment of human FSGS.

YAP, a key effector of Hippo pathway, is activated by its translocation from cytoplasm to nucleus to regulate gene expression and promote tumorigenesis. Although the mechanism by which YAP is suppressed in cytoplasm has been well-studied, how the activated YAP is sequestered in the nucleus remains unknown. Here, we demonstrate that YAP is a nucleocytoplasmic shuttling protein and its nuclear export is controlled by SET1A-mediated mono-methylation of YAP at K342, which disrupts the binding of YAP to CRM1. YAP mimetic methylation knockin mice are more susceptible to colorectal tumorigenesis. Clinically, YAP K342 methylation is reversely correlated with cancer survival. Collectively, our study identifies SET1A-mediated mono-methylation at K342 as an essential regulatory mechanism for regulating YAP activity and tumorigenesis.

Long non-coding RNAs (LncRNAs) have been identified as critical regulators in a variety of cancer types. Cancer susceptibility candidate 15 (CASC15), a lncRNA located at chromosome 6p22.3, has been discovered to participate in melanoma progression and phenotype switching. Nevertheless, the roles and molecular mechanisms of CASC15 in melanoma are far from being understood.

Coelibactin is a putative non-ribosomally synthesized peptide with predicted zincophore activity and which has been implicated in antibiotic regulation in Streptomyces coelicolor A3(2). The coelibactin biosynthetic pathway contains a stereo- and regio-specific monooxygenation step catalyzed by a cytochrome P450 enzyme (CYP105N1). We have determined the X-ray crystal structure of CYP105N1 at 2.9 Å and analyzed it in the context of the bacterial CYP105 family as a whole. The crystal structure reveals a channel between the α-helical domain and the β-sheet domain exposing the heme pocket and the long helix I to the solvent. This wide-open conformation of CYP105N1 may be related to the bulky substrate coelibactin. The ligand-free CYP105N1 structure has enough room in the substrate access channel to allow the coelibactin to enter into the active site. Analysis of typical siderophore ligands suggests that CYP105N1 may produce derivatives of coelibactin, which would then be able to chelate the zinc divalent cation.

Type 1 interferons (IFN) protect the host against viruses by engaging a cognate receptor (consisting of IFNAR1/IFNAR2 chains) and inducing downstream signaling and gene expression. However, inflammatory stimuli can trigger IFNAR1 ubiquitination and downregulation thereby attenuating IFN effects in vitro. The significance of this paradoxical regulation is unknown. Presented here results demonstrate that inability to stimulate IFNAR1 ubiquitination in the Ifnar1(SA) knock-in mice renders them highly susceptible to numerous inflammatory syndromes including acute and chronic pancreatitis, and autoimmune and toxic hepatitis. Ifnar1(SA) mice (or their bone marrow-receiving wild type animals) display persistent immune infiltration of inflamed tissues, extensive damage and gravely inadequate tissue regeneration. Pharmacologic stimulation of IFNAR1 ubiquitination is protective against from toxic hepatitis and fulminant generalized inflammation in wild type but not Ifnar1(SA) mice. These results suggest that endogenous mechanisms that trigger IFNAR1 ubiquitination for limiting the inflammation-induced tissue damage can be purposely mimicked for therapeutic benefits.

Alzheimer's disease (AD) is the most common and complex neurodegenerative disease in the elderly individuals. Recently, genome-wide association studies (GWAS) have been used to investigate AD pathogenesis. These GWAS have yielded important new insights into the genetic mechanisms of AD. However, these newly identified AD susceptibility loci exert only very small risk effects and cannot fully explain the underlying AD genetic risk. We hypothesize that combining the findings from different AD GWAS may have greater power than genetic analysis alone. To identify new AD risk factors, we integrated findings from 3 previous large-scale AD GWAS (n = 14,138) using a gene-based meta-analysis and subsequently conducted a pathway analysis using the kyoto encyclopedia of genes and genomes and gene ontology databases. Interestingly, we not only confirmed previous findings, but also highlighted, for the first time, the involvement of cardiovascular disease-related pathways in AD. Our results provided the clues as to the link between these diseases using pathway analysis methods. We believe that these findings will be very useful for future genetic studies of AD.

Melittin is a water-soluble toxic peptide derived from the venom of the bee. Although many studies show the anti-tumor activity of melittin in human cancer including glioma cells, the underlying mechanisms remain elusive. Here the effect of melittin on human hepatocelluar carcinoma HepG2 cell proliferation in vitro and further mechanisms was investigated. We found melittin could inhibit cell proliferation in vitro using Flow cytometry and MTT method. Besides, we discovered that melittin significantly downregulated the expressions of CyclinD1 and CDK4. Results of western Blot and Real-time PCR analysis indicated that melittin was capable to upregulate the expression of PTEN and attenuate histone deacetylase 2 (HDAC2) expression. Further studies demonstrated that knockdown of HDAC2 completely mimicked the effects of melittin on PTEN gene expression. Conversely, it was that the potential utility of melittin on PTEN expression was reversed in cells treated with a recombinant pEGFP-C2-HDAC2 plasmid. In addition, treatment with melittin caused a downregulation of Akt phosphorylation, while overexpression of HDAC2 promoted Akt phosphorylation. These findings suggested that the inhibitory of cell growth by melittin might be led by HDAC2-mediated PTEN upregulation, Akt inactivation, and inhibition of the PI3K/Akt signaling pathways.

Naringin, the primary active compound of the traditional Chinese medicine Rhizoma drynariae, possesses many pharmacological activities. The present study is an effort to explore the anti-osteoporosis potential of naringin in vivo and in vitro. In vivo, we used ovariectomized rats to clarify the mechanisms by which naringin anti-osteoporosis. In vitro, we used osteoclasts to investigate naringin promotes osteoclasts apoptosis. Naringin was effective at enhancing BMD, trabecular thickness, bone mineralization, and mechanical strength in a dose-dependent manner. The result of RT-PCR analysis revealed that naringin down-regulated the mRNA expression levels of BCL-2 and up-regulated BAX, caspase-3 and cytochrome C. In addition, naringin significantly reduced the bone resorption area in vitro. These findings suggest that naringin promotes the apoptosis of osteoclasts by regulating the activity of the mitochondrial apoptosis pathway and prevents OVX-induced osteoporosis in rats.

Myostatin, a member of the TGF-β superfamily, has been shown to act as a negative regulator of myogenesis. Although its role in myogenesis has been clearly documented through genetic analysis, few gene cascades that respond to myostatin signaling and regulate myogenesis have been characterized, especially in avian species. In a previous study, we screened for such genes in chicken fetal myoblasts (CFMs) using the differential display PCR method and found that cardiac ankyrin repeat protein (CARP) was downregulated by myostatin and specifically expressed in chicken skeletal muscle. However, little is known about the potential functions of CARP in chicken skeletal myogenesis. In this study, the expression patterns of chicken CARP and the possible function of this gene in skeletal muscle growth were characterized. Our data showed that CARP was predominantly expressed in postnatal skeletal muscle, and its expression increased during myogenic differentiation in CFM cells. When CARP was overexpressed, CFM cell growth was enhanced by accelerating the cell cycle at the G1 to S phase transition and increasing cyclin D1 expression. CARP knockdown had the opposite effect: while myoblasts underwent differentiation, knockdown of CARP expression induced extensive cell death, suppressed the formation of myotubes, and markedly decreased the expression of differentiation-related genes such as myosin heavy chain (MHC), myoD, and caveolin-3. Our findings indicate that CARP may play a key role in the myostatin signaling cascade that governs chicken skeletal myogenesis through promoting proliferation and avoiding apoptosis during CFM cell differentiation.

Chrysotile asbestos is closely associated with excess mortality from pulmonary diseases such as lung cancer, mesothelioma, and asbestosis. Although multiple mechanisms in which chrysotile asbestos fibers induce pulmonary disease have been identified, the role of autophagy in human lung epithelial cells has not been examined. In this study, we evaluated whether chrysotile asbestos induces autophagy in A549 human lung epithelial cells and then analyzed the possible underlying molecular mechanism. Chrysotile asbestos induced autophagy in A549 cells based on a series of biochemical and microscopic autophagy markers. We observed that asbestos increased expression of A549 cell microtubule-associated protein 1 light chain 3 (LC3-II), an autophagy marker, in conjunction with dephosphorylation of phospho-AKT, phospho-mTOR, and phospho-p70S6K. Notably, AKT1/AKT2 double-knockout murine embryonic fibroblasts (MEFs) had negligible asbestos-induced LC3-II expression, supporting a crucial role for AKT signaling. Chrysotile asbestos also led to the phosphorylation/activation of Jun N-terminal kinase (JNK) and p38 MAPK. Pharmacologic inhibition of JNK, but not p38 MAPK, dramatically inhibited the protein expression of LC3-II. Moreover, JNK2(-/-) MEFs but not JNK1(-/-) MEFs blocked LC3-II levels induced by chrysotile asbestos. In addition, N-acetylcysteine, an antioxidant, attenuated chrysotile asbestos-induced dephosphorylation of P-AKT and completely abolished phosphorylation/activation of JNK. Finally, we demonstrated that chrysotile asbestos-induced apoptosis was not affected by the presence of the autophagy inhibitor 3-methyladenine or autophagy-related gene 5 siRNA, indicating that the chrysotile asbestos-induced autophagy may be adaptive rather than prosurvival. Our findings demonstrate that AKT/mTOR and JNK2 signaling pathways are required for chrysotile asbestos-induced autophagy. These data provide a mechanistic basis for possible future clinical applications targeting these signaling pathways in the management of asbestos-induced lung disease.

Our work explores the relationship between G protein-coupled receptor kinase-5 (GRK5) single nucleotide polymorphisms and Alzheimer's disease risk. We confirmed that GRK5 translocates from the cellular membrane to the cytosol in the hippocampus of Alzheimer's disease mice and that GRK5 deficiency promotes tau hyperphosphorylation, a hallmark of Alzheimer's disease pathology. Our results indicate that one functional variant, or mutant, of GRK5 (GRK5-Gln41Leu) decreased GRK5 translocation from the membrane to the cytoplasm and reduced tau hyperphosphorylation, whereas, another GRK5 mutant (GRK5-Arg304His) increased GRK5 translocation to the cytoplasm and promoted tau hyperphosphorylation. In addition, case-control studies revealed that GRK5-Gln41Leu is associated with a lower risk of late-onset Alzheimer's disease. Our findings suggest that the GRK5-Gln41Leu mutant may resist tau hyperphosphorylation by promoting GRK5 membrane stability and, in effect, may contribute to lower Alzheimer's disease risk.