Studies of SARS coronavirus (SARS-CoV)-the causative agent of severe acute respiratory syndrome (SARS)-have been hampered by its high transmission rate and the pathogenicity of this virus. To permit analysis of the host range and entry mechanism of SARS-CoV, we incorporated the humanized SARS-CoV spike (S) glycoprotein into HIV particles to generate a highly infectious SARS-CoV pseudotyped virus. The infection on Vero E6-a permissive cell line to SARS-CoV-could be neutralized by sera from convalescent SARS patients, and the entry was a pH-dependent process. With these highly infectious SARS-CoV pseudotypes, several cell lines derived from various tissues were revealed as susceptible to SARS-CoV, which were highly corresponding to the expression pattern of virus's receptor angiotensin-converting enzyme 2 (ACE2). In addition, we also demonstrated angiotensin 1 converting enzyme (ACE)-the homologue of ACE2 could not function as a receptor for SARS-CoV.

Glioma glutamate release has been shown to promote the growth of glioma cells and induce neuronal injuries from epilepsy to neuronal death. However, potential counteractions from normal astrocytes against glioma glutamate release have not been fully evaluated. In this study, we investigated the glutamate/glutamine cycling between glioma cells and astrocytes and their impact on neuronal function. Co-cultures of glioma cells with astrocytes (CGA) in direct contact were established under different mix ratio of astrocyte/glioma. Culture medium conditioned in these CGAs were sampled for HPLC measurement, for neuronal ratiometric calcium imaging, and for neuronal survival assay. We found: (1) High levels of glutaminase expression in glioma cells, but not in astrocytes, glutaminase enables glioma cells to release large amount of glutamate in the presence of glutamine. (2) Glutamate levels in CGAs were directly determined by the astrocyte/glioma ratios, indicating a balance between glioma glutamate release and astrocyte glutamate uptake. (3) Culture media from CGAs of higher glioma/astrocyte ratios induced stronger neuronal Ca(2+) response and more severe neuronal death. (4) Co-culturing with astrocytes significantly reduced the growth rate of glioma cells. These results indicate that normal astrocytes in the brain play pivotal roles in glioma growth inhibition and in reducing neuronal injuries from glioma glutamate release. However, as tumor growth, the protective role of astrocytes gradually succumb to glioma cells.

Growing evidence suggests that hypoxia-inducible factor-α (HIF-1α) plays an important role in the progression of allergic airway inflammation and remodeling. However, the biochemical mechanisms leading to the activation of HIF-1α and the effects of HIF-1α on the expression of growth factors, including vascular endothelial growth factor (VEGF), transforming growth factor-β1 (TGF-β1), and fibroblast growth factor-2 (FGF-2), in allergic nasal inflammation are not clear. We examined the relationship between HIF-1α activation and production of VEGF, TGF-β1, and FGF-2 in primary cultured nasal epithelial cells (NECs) after stimulation with house dust mite (HDM) extract. Moreover, we evaluated the importance of phosphoinositide3-kinase(PI3K)/Akt signaling in HDM-induced production of these growth factors in vitro and in the nasal mucosa of a murine model of allergic rhinitis (AR). Our results indicate HDM extract induced the expression of VEGF, TGF-β1, and FGF-2 by activating the PI3K/Akt/HIF-1α pathway in human primary cultured NECs and in the nasal mucosa of a murine model. HIF-1α regulated the expression of VEGF, TGF-β1, and FGF-2 in the nasal mucosa through direct and indirect pathways, which suggested that targeting the HIF-1α pathway could be a novel therapeutic approach for reducing nasal airway inflammation and remodeling in AR.

Oxidative stress-mediated cell death in cardiomyocytes reportedly plays an important role in many cardiac pathologies. Our previous report demonstrated that mitochondrial SIRT3 plays an essential role in mediating cell survival in cardiac myocytes, and that resveratrol protects cardiomyocytes from oxidative stress-induced apoptosis by activating SIRT3. However, the exact mechanism by which SIRT3 prevents oxidative stress remains unknown. Here, we show that exposure of H9c2 cells to 50 μM H(2)O(2) for 6h caused a significant increase in cell death and the down-regulation of SIRT3. Reactive oxygen species (ROS)-mediated NF-κB activation was involved in this SIRT3 down-regulation. The SIRT3 activator, resveratrol, which is considered an important antioxidant, protected against H(2)O(2)-induced cell death, whereas the SIRT inhibitor, nicotinamide, enhanced cell death. Moreover, resveratrol negatively regulated H(2)O(2)-induced NF-κB activation, whereas nicotinamide enhanced H(2)O(2)-induced NF-κB activation. We also found that SOD2, Bcl-2 and Bax, the downstream genes of NF-κB, were involved in this pathological process. These results suggest that SIRT3 protects cardiomyocytes exposed to oxidative stress from apoptosis via a mechanism that may involve the NF-κB pathway.

Histidine decarboxylase gene (HDC) encodes histidine decarboxylase which is the crucial enzyme for the biosynthesis of histidine. Studies have shown that histamine is likely to be involved in the regulation of reproduction system. To find the possible correlation between HDC gene and AANM (age at natural menopause), we selected 265 postmenopausal women from 131 nuclear families and performed a transmission disequilibrium test. Significant within-family associations with AANM for SNP rs854163 and SNP rs854158 of HDC gene were observed (P values=0.0018 and 0.0197, respectively). After 1000 permutations, SNP rs854163 still remained significant within-family association with AANM. Consistently, we also detected a significant within-family association between haplotype block 2 (defined by SNP rs854163 and rs860526) and AANM in the haplotype analyses (P value=0.0397). Our results suggest that the HDC gene polymorphisms are significantly associated with AANM in Caucasian women.

Dicot Dicer-like (DCL) enzymes operate preferably on GC rich regions when producing small interfering (si)RNA and micro (mi)RNA. This GC bias, however, is not generic in monocot miRNA productions. From wild Dactylis glomerata naturally infected by Cocksfoot streak potyvirus (CSV), CSV-siRNAs had a greater GC% than the virus genome, indicating that GC rich regions were also preferred by the grass DCLs. This supports the notion that GC preference is an ancient feature for plant DCLs, and suggests that monocot miRNA genes might have evolved to a high GC% resulting in GC bias being not detectable during mature miRNA production.

Developmental dysplasia of the hip (DDH) is a common musculoskeletal disorder characterized by a mismatch between acetabulum and femoral head. Mechanical force plays an important role during the occurrence and development of abnormities in acetabulum and femoral head. In this study, we established a mechanical force model named cyclic compressive stress (Ccs). To analyze the effect of Ccs on DDH, we detected special genes in chondrocytes and osteoblasts. Results showed that Ccs downregulated chondrogenesis of ADTC5 in a concentration-dependent manner. Moreover, the mRNA level of Scinderin (Scin) considerably increased. We established lentivirus-SCIN(GV144-SCIN) to transfect hBMSCs, which were treated with different Ccs levels (0.25 Hz*5 cm, 0.5 Hz*5 cm, and 1 Hz*10 cm); the result showed that overexpression of Scin upregulated osteogenesis and osteoclastogenesis. By contrast, expression of chondrocyte-specific genes, including ACAN, COL-2A, and Sox9, decreased. Further molecular investigation demonstrated that Scin promoted osteogenesis and osteoclastogenesis through activation of the p-Smad1/5/8, NF-κB, and MAPK P38 signaling pathways, as well as stimulated the expression of key osteoclast transcriptional factors NFATc1 and c-Fos. Moreover, Scin-induced osteogenesis outweighed osteoclastogenesis in defective femur in vivo. The results of the analysis of Micro-CT confirmed these findings. Overall, Ccs influenced the development of DDH by promoting osteogenesis and cartilage degradation. In addition, Scin played a vital role in the development of DDH.

The E3 ubiquitin (Ub) ligase gp78 plays an important role in endoplasmic reticulum (ER)-associated degradation (ERAD) and regulation of lipid biogenesis. Although a variety of substrates of gp78 have been described, the regulation of the degradation of gp78 itself remains poorly understood. To address this problem, we used co-immunoprecipitation-coupled liquid chromatography-tandem mass spectrometry (Co-IP/LC-MS/MS) to identify novel proteins interacting with gp78. One of the proteins identified in this study is the deubiquitylating (DUB) enzyme USP34 (Ub-specific protease 34). We demonstrate that knockdown of USP34 facilitates proteasomal degradation of gp78 and consequently impairs the function of gp78 in regulating lipid droplet formation. This study unveils a previously unknown function of USP34 in regulating the metabolic stability of gp78 and adds to our understanding of the relevance of partnering of DUBs and E3s in regulation of protein ubiquitylation.

Osmotic stress is one of the main stresses seriously affects the growth and development of plants. Hydrogen sulfide (H2S) emerges as the third gaseous signal molecule to involve in the complex network of signaling events. Phospholipase Dδ (PLDδ), as signal enzyme, responds to many biotic or abiotic stress responses. In this study, the functions and the relationship of PLDδ and H2S in stomatal closure induced by osmotic stress were explored. Using the seedlings of ecotype (WT), PLDδ deficient mutant (pldδ), L-cysteine desulfhydrase (LCD) deficient mutant (lcd) and pldδlcd double mutant as materials, the Real-time quantitative PCR (RT-qPCR) and the stomatal aperture were analyzed. Osmotic stress induced the expressions of PLDδ and LCD. The H2S content and the activities of PLD and LCD ascended in WT under osmotic stress. The phenotypes of pldδ, lcd and pldδlcd were more sensitive to osmotic stress than WT. Compared with pldδ, the stomatal of lcd showed lower sensitivity to osmotic stress, and the stomatal aperture of pldδlcd was similar to that of lcd. Simultaneous application of PA and NaHS resulted in tighter closure of stomatal than application of either PA or NaHS alone. These results suggested that osmotic stress-triggered stomatal closure requires PLDδ and H2S in A. thaliana. LCD acted downstream of PLDδ to regulate the stomatal closure induced by osmotic stress.

Previous studies suggest that genistein protects liver from acetaminophen (APAP)-induced injury, however, the detailed mechanism of the process is still incompletely. Therefore, present study was to investigate the potential mechanism of the genistein mediated protection against APAP-induced hepatotoxicity. As shown, supplementation with 150 mg/kg genistein greatly alleviated the increase in serum alanine aminotransferase (ALT) activity, aspartate aminotransferase (AST) activity, hepatic malondialdehyde (MDA) contents, and reversed the decrease in hepatic GSH levels in response to overdose APAP. At the same time, hepatic SIRT1 protein and activity were markedly upregulated in mouse receiving genistein. However, the amelioration was almost abolished by the knockdown of hepatic SIRT1 expression using lentivirus carrying specific shRNA targeting SIRT1. These results were further validated by histopathology examination. Moreover, depletion of hepatic SIRT1 prevented the accumulation of Nrf2 in nucleus and the upregulation of the antioxidant gene expression in the presence of genistein and/or APAP. Concomitantly, the induced mRNA expression of UDP-glucuronosyltransferases (UGTs) by genistein was largely dependent on the SIRT1 expression and activity. Together, our results support the notion that the strong elevation of SIRT1 expression and activity may represent a potential mechanism of protection against APAP-induced liver injury by genistein.

A growing number of studies suggest that synovitis plays an important role in the pathogenesis and progression of osteoarthritis (OA). As a negative mediator of the nuclear factor-kappa B (NF-κB) signaling pathway, the zinc finger protein A20 has significant anti-inflammatory properties. In this study, the differential expression of A20 was investigated at the mRNA and protein levels in human normal OA fibroblast-like synoviocytes (FLSs) and normal FLSs pretreated with TNF-α. We then measured the activation of the NF-κB pathway and expression of pro-inflammatory cytokines in the above three groups by western blotting, a human cytokine array and ELISA. We found that TNF-α activated the NF-κB pathway, increased the expression of the pro-inflammatory cytokines IL-6 and IL-8, and A20 expression in human normal FLSs. However, the role of A20 in FLSs was unclear. To clarify this, we investigated the effect of A20 overexpression in human normal FLSs. The results indicate that A20 inhibits the NF-κB signaling pathway activation and OA-associated pro-inflammatory cytokines release. The results of this study indicate that A20 has anti-inflammatory effects in FLSs, which makes it a potential target for OA synovitis treatment.

Prostate cancer (PCa) refers to malignant tumors derived from prostate epithelial cells, whose morbidity and mortality rates have been increasing every year. Although new drugs for treating prostate cancer continue to emerge, the unclear mechanism underlying drug targets limits this therapy, thereby constraining identification of effective therapeutic targets. Although GDP dissociation inhibitor 2(GDI2) is highly expressed and closely associated with occurrence and development of many tumors, its role in prostate cancer remains unclear. In this study, we investigated the role of GDI2 and elucidated its underlying mechanism of action in prostate cancer. Moreover, we screened chemotherapeutic drugs that affect GDI2 expression with a view of identifying novel targets for diagnosis and treatment of prostate cancer.

Alzheimer's disease (AD) is a chronic neurodegenerative disease featured by progressive memory loss and cognitive dysfunction. Long non-coding RNAs are recently demonstrated as important regulatory molecules in neurodegenerative diseases. This study explored regulatory role of lncRNA small nucleolar RNA host gene 1 (SNHG1) in the neuronal cell injury induced by Aβ25-35. Our results showed that Aβ25-35 inhibited cell viability, induced cell apoptosis and increased the expression of SNHG1 in SH-SY5Y and human primary neuron (HPN) cells. Knockdown of SNHG1 partially reversed the effects of Aβ25-35 treatment on cell viability, cell apoptosis, mitochondrial membrane potential (MMP), caspase-3 activity, and apoptosis signaling-related protein levels in SH-SY5Y and HPN cells. The bioinformatics analysis and luciferase reporter assay showed that SNHG1 functioned as competing endogenous RNA (ceRNA) for miR-137, and pre-treatment with SNHG1 siRNA increased cell viability, suppressed cell apoptosis, increased MMP, decreased caspase-3 activity and caused a decrease in the protein levels of cytochrome C and cleaved caspase-3 and an increase in Bcl-2 protein level in the Aβ25-35-treated SH-SY5Y and HPN cells, which was significantly attenuated by the presence of miR-137 mimics. Moreover, miR-137 negatively regulated the expression of kringle containing transmembrane protein 1 (KREMEN1) via targeting its 3' untranslated region, and knockdown of SNHG1 also suppressed KREMEN1 in SH-SY5Y and HPN cells. Overexpression of KREMEN1 impaired the neuronal protective effects of SNHG1 knockdown in the Aβ25-35-treated SH-SY5Y and HPN cells. In summary, our result indicated that knockdown of SNHG1 exerted its neuronal protective effects via repressing KRENEN1 by acting as a ceRNA for miR-137 in the in vitro cell model of AD.

Here we explored the anti-oxidative and cytoprotective potentials of escin, a natural triterpene-saponin, against hydrogen peroxide (H2O2) in retinal pigment epithelium (RPE) cells. We showed that escin remarkably attenuated H2O2-induced death and apoptosis of established (ARPE-19) and primary murine RPE cells. Meanwhile, ROS production and lipid peroxidation by H2O2 were remarkably inhibited by escin. Escin treatment in RPE cells resulted in NF-E2-related factor 2 (Nrf2) signaling activation, evidenced by transcription of anti-oxidant-responsive element (ARE)-regulated genes, including HO-1, NQO-1 and SRXN-1. Knockdown of Nrf2 through targeted shRNAs/siRNAs alleviated escin-mediated ARE gene transcription, and almost abolished escin-mediated anti-oxidant activity and RPE cytoprotection against H2O2. Reversely, escin was more potent against H2O2 damages in Nrf2-over-expressed ARPE-19 cells. Further studies showed that escin-induced Nrf2 activation in RPE cells required AKT signaling. AKT inhibitors (LY294002 and perifosine) blocked escin-induced AKT activation, and dramatically inhibited Nrf2 phosphorylation, its cytosol accumulation and nuclear translocation in RPE cells. Escin-induced RPE cytoprotection against H2O2 was also alleviated by the AKT inhibitors. Together, these results demonstrate that escin protects RPE cells from oxidative stress possibly through activating AKT-Nrf2 signaling.

Abnormal neurogenesis occurs during embryonic development in human diabetic pregnancies and in animal models of diabetic embryopathy. Our previous studies in a mouse model of diabetic embryopathy have implicated that high glucose of maternal diabetes delays neurogenesis in the developing neuroepithelium leading to neural tube defects. However, the underlying process in high glucose-impaired neurogenesis is uncharacterized. Neurogenesis from embryonic stem (ES) cells provides a valuable model for understanding the abnormal neural lineage development under high glucose conditions. ES cells are commonly generated and maintained in high glucose (approximately 25 mM glucose). Here, the mouse ES cell line, E14, was gradually adapted to and maintained in low glucose (5 mM), and became a glucose responsive E14 (GR-E14) line. High glucose induced the endoplasmic reticulum stress marker, CHOP, in GR-E14 cells. Under low glucose conditions, the GR-E14 cells retained their pluripotency and capability to differentiate into neural lineage cells. GR-E14 cell differentiation into neural stem cells (Sox1 and nestin positive cells) was inhibited by high glucose. Neuron (Tuj1 positive cells) and glia (GFAP positive cells) differentiation from GR-E14 cells was also suppressed by high glucose. In addition, high glucose delayed GR-E14 differentiation into neural crest cells by decreasing neural crest markers, paired box 3 (Pax3) and paired box 7 (Pax7). Thus, high glucose impairs ES cell differentiation into neural lineage cells. The low glucose adapted and high glucose responsive GR-E14 cell line is a useful in vitro model for assessing the adverse effect of high glucose on the development of the central nervous system.

Currently, there are no effective therapeutic strategies targeting Kras driven cancers, and therefore, identifying new targeted therapies and overcoming drug resistance have become paramount for effective long-term cancer therapy. We have found that reducing expression of the palmitoyl transferase DHHC20 increases cell death induced by the EGFR inhibitor gefitinib in Kras and EGFR mutant cell lines, but not MCF7 cells harboring wildtype Kras. We show that the increased gefitinib sensitivity in cancer cells induced by DHHC20 inhibition is mediated directly through loss of palmitoylation on a previously identified cysteine residue in the C-terminal tail of EGFR. We utilized an EGFR point mutant in which the palmitoylated cysteine 1025 is mutated to alanine (EGFRC1025A), that results in receptor activation. Expression of the EGFR mutant alone in NIH3T3 cells does not increase sensitivity to gefitinib-induced cell death. However, when EGFRC1025A is expressed in cells expressing activated KrasG12V, EGFR inhibitor induced cell death is increased. Surprisingly, lung cancer cells harboring the EGFR inhibitor resistant mutation, T790M, become sensitive to EGFR inhibitor treatment when DHHC20 is inhibited. Finally, the small molecule, 2-bromopalmitate, which has been shown to inhibit palmitoyl transferases, acts synergistically with gefitinib to induce cell death in the gefitinib resistant cell line NCI-H1975.

Our previous studies have indicated that Bombyx mori receptor expression enhancing protein a (BmREEPa) could participate in BV invasion in vivo and in vitro, however, the mechanism is still unclear. In this study, we screened BmREEPa interacting protein through co-immunoprecipitation and finally identified a membrane protein, Bombyx mori patched domain containing protein (BmPtchd, KR338939), which contains receptor activity. Further studies showed that BmPtchd, BmREEPa and Glycoprotein 64 could form a protein complex and the expression level of BmREEPa and BmPtchd could be affected by cellular cholesterol level. These findings may provide an important basis for explaining the invasion mechanism of Bombyx mori Nucleopolyhedrovirus budded virus.

Spastic paraplegia 20 methylation was characterized in gastric cancer in our previous study. However, its mechanism remains unknown. Cell proliferation, colony formation, flow cytometry, wound healing, in vitro Transwell assays and in vivo xenografts were performed. A nomogram model was established to make a more accurate prognostic prediction for gastric cancer patients. Knockout of Spastic paraplegia 20 promoted gastric cancer cell proliferation, G2/M arrest in vitro and tumor growth in vivo. The EGFR/MAPK pathway was activated as a consequence of Spastic paraplegia 20 deletion. EGFR kinase or ERK1/2 inhibitors impaired Spastic paraplegia 20 knockout-induced cancer cell growth. Gastric cancer patients with poor spartin expression (72/161, 44.7%) exhibited a worse prognosis compared with the high expression group with median survival times of 16 and 54 months, respectively. The nomogram model stratified gastric cancer patients into 3 distinct prognostic groups with 3-year survival rates of 100%, 77%, and 35%. Furthermore, it had a better discrimination than the TNM staging system (C index: 0.785, AIC: 752.8708 VS. C index: 0.712; AIC: 775.1223). Methylation-induced Spastic paraplegia 20 silencing facilitates gastric cancer cell proliferation by activating the EGFR/MAPK signaling pathway. The nomogram based on spartin expression provided significantly better discrimination compared with the traditional AJCC TNM staging system and provided an individualized prediction of the survival for gastric cancer patient survival.

Hypoxia and islet inflammation are involved in β-cell failure in type 2 diabetes (T2D). Elevated plasma LPS levels have been verified in patients with T2D, and hypoxia occurs in islets of diabetic mice. Activation of inflammasomes in ischemic or hypoxic conditions was identified in various tissues. Here, we investigated whether hypoxia activates the inflammasome in β cells and the possible mechanisms involved. In mouse insulinoma cell line 6 (MIN6), hypoxia (1% O2) primes the NLRP3 inflammasome along with NF-κB signaling activation. Our results demonstrate that hypoxia can activate the NLRP3 inflammasome in LPS-primed MIN6 to result in initiating the β cell inflammatory response and cell death in vitro. Reactive oxygen species (ROS) and the thioredoxin-interacting protein (TXNIP) are up-regulated in response to hypoxia. Finally, the role of the ROS-TXNIP axis in mediating the activation of the NLRP3 inflammasome and cell death was characterized by pretreating with the ROS scavenger N-acetylcysteine (NAC) and performing TXNIP knockdown experiments in MIN6. Our data indicate for the first time that the inflammasome is involved in the inflammatory response and cell death in hypoxia-induced β cells through the ROS-TXNIP-NLRP3 axis in vitro. This provides new insight into the relationship between hypoxia and inflammation in T2D.

Treatment of cells with carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial proton gradient uncoupler, can result in mitochondrial damage and autophagy activation, which in turn eliminates the injured mitochondria in a Parkin-dependent way. How CCCP mobilizes the autophagy machinery is not fully understood. By analyzing a key autophagy step, LC3 lipidation, we examined the roles of two kinase complexes typically involved in the initiation and nucleation phases of autophagy, namely the ULK kinase complex (UKC) and the Beclin 1/Atg14 complex. We found that CCCP-induced LC3 lipidation could be independent of Beclin 1 and Atg14. In addition, deletion or knockdown of the UKC component FIP200 or Atg13 only led to a partial reduction in LC3 lipidation, indicating that UKC could be also dispensable for this step during CCCP treatment. In contrast, Atg9, which is important for transporting vesicles to early autophagosomal structure, was required for CCCP-induced LC3 lipidation. Taken together, these data suggest that CCCP-induced autophagy and mitophagy depends more critically on Atg9 vesicles than on UKC and Beclin 1/Atg14 complex.