DNA self-assembly nanoflower reverse P-glycoprotein mediated drug resistance in chronic myelogenous leukemia therapy.

Introduction: Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder caused by the BCR-ABL chimeric tyrosine kinase. Vincristine (VCR) is widely used in leukemia therapy but is hindered by multidrug resistance (MDR). Methods: We prepared DNA nanoflower via self-assembly for the delivery of VCR and P-glycoprotein small interfering RNA (P-gp siRNA). Results and Discussion: The as-prepared nanoflower had a floriform shape with high loading efficiency of VCR (80%). Furthermore, the nanoflower could deliver VCR and P-gp siRNA into MDR CML cells and induce potent cytotoxicity both in vitro and in vivo, thus overcoming MDR of CML. Overall, this nanoflower is a promising tool for resistant CML therapy.

Succession of endophytic bacterial community and its contribution to cinnamon oil production during cinnamon shade-drying process.

Cinnamon oil is a blend of secondary metabolites and is widely used as spice. Endophytic bacteria are always related to the secondary metabolites production. However, the potential of endophytic bacteria communities for cinnamon oil production during cinnamon shade-drying process is still not clear. In this study, we investigated the composition and metabolic function of endophytic bacterial community during 80-day shade-drying process. The temporal dynamics of essential oil content and its dominant constituents were analyzed. The succession of endophytic bacterial community from d0 to d80 was identified. The influence of endophytic bacterial community evolution on cinnamon oil is significant positive. Predictive functional analysis indicated that shade-drying process was rich in Saccharopolyspora that produce enzymes for the conversion of phenylalanine to cinnamaldehyde. These findings enhance our understanding of the functional bacterial genera and functional genes involved in the production of cinnamon oil during cinnamon shade-drying process.

Synthesis, Herbicidal Activity and Toxicity Evaluation of Secondary Ammonium Salts from Turpentine Oil for Sustainable Weed Control.

Three series of secondary ammonium chloride from turpentine were synthesized and evaluated as botanical herbicides. The preemergence herbicidal activities against ryegrass (Loliun multiflorum) and barnyard grass (Echinochloa crus-galli) were investigated using water as the only solvent. Their toxicity was evaluated by cytotoxicity assays. Preliminary results demonstrated that the herbicidal performance of the prepared salts was similar or much higher than that of corresponding secondary amines and even commercial herbicide glyphosate. Promisingly, compound 14e containing a cyclohexyl-substituted p-menthene skeleton with an IC50 value of 0.0014 mM against root growth of ryegrass showed 39-fold higher herbicidal activity than glyphosate. Besides, this compound was found to be nontoxic to human and animal cells, indicating the potential application as a water-soluble herbicide for ryegrass control.

Synthesis and biological activity of pyridine acylhydrazone derivatives of isopimaric acid.

Pyridine acylhydrazone derivatives of isopimaric acid were synthesized and characterized. The minimum inhibitory concentrations of the compounds against five bacteria were determined and most of the compounds displayed some degree of antibacterial activity. The results showed that antimicrobial activity against Streptococcus pneumoniae improved when halogen atoms were introduced into the isopimaric acid, especially when one bromine atom was introduced in the para-position of isopimaric acid. Compound isopimaric acid (5-bromo pyridine-2-formaldehyde) acylhydrazone exhibited a significant antitumorial activity against hepatocarcioma cells (HepG-2) and breast cancer cells (MDA-MB-231), with inhibition degrees of 74.21% and 70.39%, respectively, at 100 µM.[Formula: see text].

Chemokine (C-X-C Motif) Ligand 4 Is a Restrictor of Respiratory Syncytial Virus Infection and an Indicator of Clinical Severity.

Rationale: Respiratory syncytial virus (RSV) is the leading cause of childhood respiratory infections worldwide; however, no vaccine is available, and treatment options are limited. Identification of host factors pivotal to viral replication may inform the development of novel therapies, prophylaxes, or diagnoses.Objectives: To identify host factors involved in RSV replication and to evaluate their potential for disease management.Methods: A gain-of-function screening was performed on the basis of a genome-wide human complementary DNA library screen for host factors involved in RSV replication. The antiviral mechanism of CXCL4 (chemokine [C-X-C motif] ligand 4) was analyzed. Its clinical role was evaluated via nasopharyngeal aspirates and plasma samples from patients with RSV infection and different disease severities.Measurements and Main Results: Forty-nine host factors restricting RSV replication were identified by gain-of-function screening, with CXCL4 showing the strongest antiviral effect, which was secretion dependent. CXCL4 blocked viral attachment through binding to the RSV main receptor heparan sulfate, instead of through interacting with RSV surface proteins. Intranasal pretreatment with CXCL4 alleviated inflammation in RSV-infected mice, as shown by decreased concentrations of tumor necrosis factor and viral load in BAL fluid samples as well as by viral nucleocapsid protein histological staining in lungs. Compared with non-RSV infections, RSV infections induced elevated CXCL4 concentrations both in plasma and airway samples from mice and pediatric patients. The airway CXCL4 concentration was correlated with viral load and disease severity in patients (P < 0.001).Conclusions: Our results suggest that CXCL4 is an RSV restriction factor that can block viral entry and serve as an indicator of clinical severity in RSV infections.

Suppression of Esophageal Cancer Stem-like Cells by SNX-2112 Is Enhanced by STAT3 Silencing.

Many studies have demonstrated that cancer stem cells (CSCs) or tumor-initiating cells (TICs) are responsible for tumor cell proliferation, chemotherapy resistance, metastasis, and relapse in various cancers. We, and others, have previously shown that the signal transducer and activator of transcription 3 (STAT3) signaling pathway is responsible for CSCs and TICs growth. Recent reports have indicated that the heat shock protein 90 (Hsp90) is also essential for the survival of CSCs and TICs. SNX-2112 is an Hsp90 inhibitor. However, it remains unclear whether proliferation of esophageal cancer stem-like cells (ECSLCs) is suppressed by SNX-2112 with knockdown of STAT3 (shSTAT3). Here, we explored the association between SNX-2112 with shSTAT3 and the suppression of ECSLCs growth. We found that the expression level of both STAT3 and p-STAT3 was higher in clinical esophageal cancer tissue than in the adjacent normal tissue, using western blot and qPCR analysis. Furthermore, differential expression analysis demonstrated that STAT3 was overexpressed in clinical specimens. We demonstrated that SNX-2112 inhibited cancer cell proliferation, decreased ABCB1 and ABCG2 gene expression levels and reduced the colony formation capacity of ECSLCs, which was enhanced by STAT3 silencing. Flow cytometry analysis revealed that the combination of SNX-2112 and shSTAT3 significantly induced apoptosis and cell cycle arrest at G2/M phase in ECSLCs. Levels of proliferation pathway proteins, including p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) which were also client proteins of Hsp90, were also reduced. In addition, SNX-2112 with shSTAT3 inhibited the proliferation of ECSLCs in vivo. Finally, STAT3 overexpression eliminated the apoptotic and antiproliferative effects of SNX-2112 on ECSLCs. Hence, these results provide a rationale for the therapeutic potential of the combination of SNX-2112 with shSTAT3 in esophageal cancer, and may indicate new targets for clinical intervention in human cancer.

A Novel DT40 Antibody Library for the Generation of Monoclonal Antibodies.

Early etiological diagnosis is very important for the control of sudden viral infections, and requires antibodies with both high sensitivity and high specificity. Traditional antibody preparation methods have limitations, such as a long and arduous cycle, complicated operation, and high expenses. A chicken lymphoma cell line, DT40, is known to produce IgM-type antibodies and undergo gene conversion and somatic mutation in the variable region of the immunoglobulin gene during culture. Here, the DT40 cell line was developed to produce antibody libraries and prepare antibody rapidly in vitro. Since hypermutation in DT40 cells was regulated by the activation-induced cytidine deaminase (AID) gene, AID expression needs to be controlled to either fix the Ig sequence by stopping mutation or improve affinity by resuming mutation after the antibodies have been selected. In this study, we generated a novel AID-inducible DT40 cell line (DT40-H7), in which the endogenous AID gene was knocked out using the CRISPR/Cas9 genome editing system, and an inducible AID gene, based on the Tet-Off expression system, was stably transfected. AID expression was controlled in DT40-H7 cells in a simple and efficient manner; gene conversion and point mutations were observed only when AID was expressed. Using the antibody library generated from this cell line, we successfully obtained monoclonal antibodies against the NS1 protein of Zika virus. The DT40-H7 cell line represents a useful tool for the selection and evolution of antibodies and may also be a powerful tool for the rapid selection and generation of diagnostic antibodies for emerging infectious diseases.

Transduction with Lentiviral Vectors Altered the Expression Profile of Host MicroRNAs.

RNA interference (RNAi) is widely used in gene knockdown analysis and as a tool to screen host genes involved in viral infection. Owing to the limitations of transducing cells with synthetic small interfering RNAs (siRNAs), lentiviral short hairpin RNA (shRNA) vectors are more widely used. However, we found that stable transduction with lentiviral shRNA vectors inhibited hepatitis C virus (HCV) propagation in human hepatoma cells. We found by microRNA (miRNA) microarray analysis that this inhibition was induced by the alteration of host miRNA expression. In addition to one miRNA (miR-196b-5p) previously reported to be involved in HCV infection, other miRNAs (miR-216a-5p, -216b-5p, 217, and -30b-5p) were found to influence HCV infection in this study. Further studies suggested that this effect was independent of the transcription of shRNAs. The lentiviral vector itself and the integration site of the lentiviral vector might determine the change in miRNA expression. Moreover, the upregulation of JUN contributed to the dysregulation of miR-216a-5p, -216b-5p, and -217 in stably transduced cells. Although the changes in miRNA expression were beneficial for inhibiting HCV infection in our study, this off-target effect should be considered when transduction with lentiviral vectors is performed for other purposes, especially in therapy.IMPORTANCE We found that stable transduction with lentiviral shRNA was able to nonspecifically inhibit HCV infection by the dysregulation of host miRNAs. Previous studies showed that the overexpression of shRNAs oversaturated the host miRNA pathways to inhibit HCV infection. In contrast, the miRNA machinery was not affected in our study. Knockout studies suggested that the nonspecific effect was independent of the transcription of shRNAs. The lentiviral vector itself and the integration sites in the host genome determined the changes in miRNAs. Stable transduction with lentiviral vectors was able to increase the expression of JUN, which in turn upregulated miR-216a-5p, miR-216b-5p, and miR-217. miR-216a-5p and miR-216b-5p might inhibit HCV by suppressing the host autophagic machinery. Our study suggested a novel nonspecific effect of lentiviral vectors, and this side effect should be considered when transduction with lentiviral vectors is performed for other purposes, especially in therapy.

T59, a New Compound Reconstructed from Curcumin, Induces Cell Apoptosis through Reactive Oxygen Species Activation in Human Lung Cancer Cells.

Curcumin is acknowledged for its antioxidant, anti-inflammatory, anti-cancer, and wound-healing properties. However, the biological activity and the molecular mechanisms of T59, which is a new derivative of curcumin, are not fully understood. The present study was aimed to determine the cytoxicity role of T59 in human lung cancer and the molecular mechanisms. Cytotoxicity and cell apoptosis effects induced by T59 were determined by MTT, AO staining, Annexin V, and JC-1. Compared with curcumin, T59 exerted more effective cytotoxicity and cell apoptosis effects in A549 and H1975. With the decreasing level of the mitochondrion membrane potential, the generation of reactive oxygen species (ROS) was increased and induced by T59. Furthermore, the expressions of cleaved-caspase-3 and Bax were increased, which were reversed by NAC mainly through the PI3K/AKT signaling pathway. Our results suggested that T59 has the potential for further investigation and study to act as an anti-cancer therapeutic against human lung cancer.

Cyclophilin A-regulated ubiquitination is critical for RIG-I-mediated antiviral immune responses.

RIG-I is a key cytosolic pattern recognition receptor that interacts with MAVS to induce type I interferons (IFNs) against RNA virus infection. In this study, we found that cyclophilin A (CypA), a peptidyl-prolyl cis/trans isomerase, functioned as a critical positive regulator of RIG-I-mediated antiviral immune responses. Deficiency of CypA impaired RIG-I-mediated type I IFN production and promoted viral replication in human cells and mice. Upon Sendai virus infection, CypA increased the interaction between RIG-I and its E3 ubiquitin ligase TRIM25, leading to enhanced TRIM25-mediated K63-linked ubiquitination of RIG-I that facilitated recruitment of RIG-I to MAVS. In addition, CypA and TRIM25 competitively interacted with MAVS, thereby inhibiting TRIM25-induced K48-linked ubiquitination of MAVS. Taken together, our findings reveal an essential role of CypA in boosting RIG-I-mediated antiviral immune responses by controlling the ubiquitination of RIG-I and MAVS.

Crystal structure of 2C helicase from enterovirus 71.

Enterovirus 71 (EV71) is the major pathogen responsible for outbreaks of hand, foot, and mouth disease. EV71 nonstructural protein 2C participates in many critical events throughout the virus life cycle; however, its precise role is not fully understood. Lack of a high-resolution structure made it difficult to elucidate 2C activity and prevented inhibitor development. We report the 2.5 Å-resolution crystal structure of the soluble part of EV71 2C, containing an adenosine triphosphatase (ATPase) domain, a cysteine-rich zinc finger with an unusual fold, and a carboxyl-terminal helical domain. Unlike other AAA+ ATPases, EV71 2C undergoes a carboxyl terminus-mediated self-oligomerization, which is dependent on a specific interaction between the carboxyl-terminal helix of one monomer and a deep pocket formed between the ATPase and the zinc finger domains of the neighboring monomer. The carboxyl terminus-mediated self-oligomerization is fundamental to 2C ATPase activity and EV71 replication. Our findings suggest a strategy for inhibition of enterovirus replication by disruption of the self-oligomerization interface of 2C.

Amphotericin B Inhibits Enterovirus 71 Replication by Impeding Viral Entry.

Enterovirus 71 (EV71) infection causes hand-foot-and-mouth disease that leads to cardiopulmonary complications and death in young children. There is thus an urgent need to find new treatments to control EV71 infection. In this study, we report potent inhibition of EV71 by a polyene antibiotic Amphotericin B. Amphotericin B profoundly diminished the expression of EV71 RNA and viral proteins in the RD cells and the HEK293 cells. As a result, EV71 production was inhibited by Amphotericin B with an EC50 (50% effective concentration) of 1.75 µM in RD cells and 0.32 µM in 293 cells. In addition to EV71, EV68 was also strongly inhibited by Amphotericin B. Results of mechanistic studies revealed that Amphotericin B targeted the early stage of EV71 infection through impairing the attachment and internalization of EV71 by host cells. As an effective anti-fungi drug, Amphotericin B thus holds the promise of formulating a novel therapeutic to treat EV71 infection.

[Protective Effects of Ginkgolide N Against Glutamate-Induced Injury in PC12 Cells].

OBJECTIVE: To study the protective effects of ginkgolide N against glutamte-induced injury in PC12 cells and its mechanisms. METHODS: The injury model was established by treating PC12 cells with glutamate, and PC12 cells were treated with different concentrations of ginkgolide N with ginkgolide B as control. The cells activity was analyzed by MTT assay. The apoptosis of PC12 cells were examined by acridine orange( AO) staining, the reactive oxygen species and mitochondrial membrane potential of PC12 cells were examined by flow cytometry. Western blot method was used to examine the expression of Cleaved Caspase-3 protein. RESULTS: Ginkgolides N of 2-8 µgmol/L inhibited PC12 cells apoptosis and ROS accumulation induced by glutamate,stabilized membrane potential of damaged PC12, and reduced the expression of Cleaved Caspase-3 protein. CONCLUSION: Ginkgolide N has a protective effect on PC12 cells injury induced by glutamate, and the mechanism may be associated with reducing ROS generation, stabilizing membrane potential and inhibiting the expression of Cleaved Caspase-3 protein.

Molecular and Cytogenetic Characterization of a Powdery Mildew-Resistant Wheat-Aegilops mutica Partial Amphiploid and Addition Line.

Aegilops mutica Boiss., a diploid species (2n = 2x = 14, TT), has been rarely studied before. In this research, a hexaploid wheat (cv. Chinese Spring)-Ae. mutica partial amphiploid and a wheat-Ae. mutica addition line were characterized by chromosome karyotyping, FISH using oligonucleotides Oligo-pTa535-1, Oligo-pSc119.2-1, and (GAA)8 as probes, and EST-based molecular markers. The results showed that the partial amphiploid strain consisted of 20 pairs of wheat chromosomes and 7 pairs of Ae. mutica chromosomes, with both wheat 7B chromosomes missing. EST-based molecular marker data suggested that the wheat-Ae. mutica addition line carries the 7T chromosome. Resistance tests indicated that both the partial amphiploid and the 7T addition line were highly resistant to powdery mildew, whereas the wheat control line Chinese Spring was highly susceptible, indicating the presence of a potentially new powdery mildew resistance gene on the Ae. mutica 7T chromosome. The karyotype, FISH patterns, and molecular markers can now be used to identify Ae. mutica chromatin in a wheat background, and the 7T addition could be used as a new powdery mildew resistance source for wheat breeding.

Hepatitis C virus core protein activates autophagy through EIF2AK3 and ATF6 UPR pathway-mediated MAP1LC3B and ATG12 expression.

HCV infection induces autophagy, but how this occurs is unclear. Here, we report the induction of autophagy by the structural HCV core protein and subsequent endoplasmic reticular (ER) stress in Huh7 hepatoma cells. During ER stress, both the EIF2AK3 and ATF6 pathways of the unfolded protein response (UPR) were activated by HCV core protein. Then, these pathways upregulated transcription factors ATF4 and DDIT3. The ERN1-XBP1 pathway was not activated. Through ATF4 in the EIF2AK3 pathway, the autophagy gene ATG12 was upregulated. DDIT3 upregulated the transcription of autophagy gene MAP1LC3B (LC3B) by directly binding to the -253 to -99 base region of the LC3B promoter, contributing to the development of autophagy. Collectively, these data suggest not only a novel role for the HCV core protein in autophagy but also offer new insight into detailed molecular mechanisms with respect to HCV-induced autophagy, specifically how downstream UPR molecules regulate key autophagic gene expression.

Crystal structure of 2A proteinase from hand, foot and mouth disease virus.

EV71 is responsible for several epidemics worldwide; however, the effective antiviral drug is unavailable to date. The 2A proteinase (2A(pro)) of EV71 presents a promising drug target due to its multiple roles in virus replication, inhibition of host protein synthesis and evasion of innate immunity. We determined the crystal structure of EV71 2A(pro) at 1.85Å resolution, revealing that the proteinase maintains a chymotrypsin-like fold. The active site is composed of the catalytic triads C110A, H21 and D39 with the geometry similar to that in other picornaviral 2A(pro), 3C(pro) and serine proteinases. The cI-to-eI2 loop at the N-terminal domain of EV71 2A(pro) adopts a highly stable conformation and contributes to the hydrophilic surface property, which are strikingly different in HRV2 2A(pro) but are similar in CVB4 2A(pro). We identified a hydrophobic motif "LLWL" followed by an acidic motif "DEE" at the C-terminus of EV71 2A(pro). The "LLWL" motif is folded into the ß-turn structure that is essential for the positioning of the acidic motif. Our structural and mutagenesis study demonstrated that both the negative charging and the correct positioning of the C-terminus are essential for EV71 replication. Deletion of the "LLWL" motif abrogated the proteolytic activity, indicating that the motif is critical for maintaining the active proteinase conformation. Our findings provide the structural and functional insights into EV71 2A(pro) and establish a framework for structure-based inhibitor design.

Validation-based insertional mutagenesis for identification of Nup214 as a host factor for EV71 replication in RD cells.

Lentiviral validation-based insertional mutagenesis (VBIM) is a sophisticated, forward genetic approach that is used for the investigation of signal transduction in mammalian cells. Using VBIM, we conducted function-based genetic screening for host genes that affect enterovirus 71 (EV71) viral replication. This included host factors that are required for the life cycle of EV71 and host restriction factors that inhibit EV71 replication. Several cell clones, resistant to EV71, were produced using EV71 infection as a selection pressure and the nuclear pore protein 214 (Nup214) was identified as a host factor required for EV71 replication. In SD2-2, the corresponding VBIM lentivirus transformed clone, the expression of endogenous Nup214 was significantly down-regulated by the reverse inserted VBIM promoter. After Cre recombinase-mediated excision of the VBIM promoter, the expression of Nup214 recovered and the clone regained sensitivity to the EV71 infection. Furthermore, over-expression of Nup214 in the cells suggested that Nup214 was promoting EV71 replication. Results of this study indicate that a successful mutagenesis strategy has been established for screening host genes related to viral replication.

Enterovirus 71 protease 2Apro targets MAVS to inhibit anti-viral type I interferon responses.

Enterovirus 71 (EV71) is the major causative pathogen of hand, foot, and mouth disease (HFMD). Its pathogenicity is not fully understood, but innate immune evasion is likely a key factor. Strategies to circumvent the initiation and effector phases of anti-viral innate immunity are well known; less well known is whether EV71 evades the signal transduction phase regulated by a sophisticated interplay of cellular and viral proteins. Here, we show that EV71 inhibits anti-viral type I interferon (IFN) responses by targeting the mitochondrial anti-viral signaling (MAVS) protein--a unique adaptor molecule activated upon retinoic acid induced gene-I (RIG-I) and melanoma differentiation associated gene (MDA-5) viral recognition receptor signaling--upstream of type I interferon production. MAVS was cleaved and released from mitochondria during EV71 infection. An in vitro cleavage assay demonstrated that the viral 2A protease (2A(pro)), but not the mutant 2A(pro) (2A(pro)-110) containing an inactivated catalytic site, cleaved MAVS. The Protease-Glo assay revealed that MAVS was cleaved at 3 residues between the proline-rich and transmembrane domains, and the resulting fragmentation effectively inactivated downstream signaling. In addition to MAVS cleavage, we found that EV71 infection also induced morphologic and functional changes to the mitochondria. The EV71 structural protein VP1 was detected on purified mitochondria, suggesting not only a novel role for mitochondria in the EV71 replication cycle but also an explanation of how EV71-derived 2A(pro) could approach MAVS. Taken together, our findings reveal a novel strategy employed by EV71 to escape host anti-viral innate immunity that complements the known EV71-mediated immune-evasion mechanisms.

The interplays between autophagy and apoptosis induced by enterovirus 71.

BACKGROUND: Enterovirus 71 (EV71) is the causative agent of human diseases with distinct severity, from mild hand, foot and mouth disease to severe neurological syndromes, such as encephalitis and meningitis. The lack of understanding of viral pathogenesis as well as lack of efficient vaccine and drugs against this virus impedes the control of EV71 infection. EV71 virus induces autophagy and apoptosis; however, the relationship between EV71-induced autophagy and apoptosis as well as the influence of autophagy and apoptosis on virus virulence remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: In this study, it was observed that the Anhui strain of EV71 induced autophagy and apoptosis in human rhabdomyosarcoma (RD-A) cells. Additionally, by either applying chemical inhibitors or knocking down single essential autophagic or apoptotic genes, inhibition of EV71 induced autophagy inhibited the apoptosis both at the autophagosome formation stage and autophagy execution stage. However, inhibition of autophagy at the stage of autophagosome and lysosome fusion promoted apoptosis. In reverse, the inhibition of EV71-induced apoptosis contributed to the conversion of microtubule-associated protein 1 light chain 3-I (LC3-I) to LC3-II and degradation of sequestosome 1 (SQSTM1/P62). Furthermore, the inhibition of autophagy in the autophagsome formation stage or apoptosis decreased the release of EV71 viral particles. CONCLUSIONS/SIGNIFICANCE: In conclusion, the results of this study not only revealed novel aspect of the interplay between autophagy and apoptosis in EV71 infection, but also provided a new insight to control EV71 infection.

Insights into the roles of cyclophilin A during influenza virus infection.

Cyclophilin A (CypA) is the main member of the immunophilin superfamily that has peptidyl-prolyl cis-trans isomerase activity. CypA participates in protein folding, cell signaling, inflammation and tumorigenesis. Further, CypA plays critical roles in the replication of several viruses. Upon influenza virus infection, CypA inhibits viral replication by interacting with the M1 protein. In addition, CypA is incorporated into the influenza virus virions. Finally, Cyclosporin A (CsA), the main inhibitor of CypA, inhibits influenza virus replication through CypA-dependent and -independent pathways. This review briefly summarizes recent advances in understanding the roles of CypA during influenza virus infection.