A human coxsackievirus B3 (CVB3), designated strain Beijing0811, was isolated from a child diagnosed with hospital-acquired infectious acute myocarditis in Beijing, China, and propagated in human rhabdomyosarcoma cells. The complete genome sequence of this virus was 7,402 nucleotides, excluding the 3′ poly(A) tail, which encoded a large polyprotein with 2,185 amino acids. This report will help us to analyze the evolutionary and epidemic characteristics of CVB3.
Hemoglobin (Hgb) is the main oxygen and carbon dioxide carrier in cells of erythroid lineage and is responsible for oxygen delivery to the respiring tissues of the body. However, Hgb is also expressed in nonerythroid cells. In the present study, the expression of Hgb in human uterine cervix carcinoma cells and its role in cervical cancer were investigated.
The expression level of Hgb in cervical cancer tissues was assessed by quantitative reverse transcriptase-PCR (qRT-PCR). We applied multiple methods, such as RT-PCR, immunoblotting, and immunohistochemical analysis, to confirm Hgb expression in cervical cancer cells. The effects of ectopic expression of Hgb and Hgb mutants on oxidative stress and cell viability were investigated by cellular reactive oxygen species (ROS) analysis and lactate dehydrogenase (LDH) array, respectively. Both Annexin V staining assay by flow cytometry and caspase-3 activity assay were used, respectively, to evaluate cell apoptosis.
qRT-PCR analysis showed that Hgb-α- (HBA1) and Hgb-β-globin (HBB) gene expression was significantly higher in cervical carcinoma than in normal cervical tissues, whereas the expression of hematopoietic transcription factors and erythrocyte specific marker genes was not increased. Immunostaining experiments confirmed the expression of Hgb in cancer cells of the uterine cervix. Hgb mRNA and protein were also detected in the human cervical carcinoma cell lines SiHa and CaSki, and Hgb expression was up-regulated by hydrogen peroxide-induced oxidative stress. Importantly, ectopic expression of wild type HBA1/HBB or HBA1, rather than mutants HBA1H88R/HBBH93R unable to bind hemo, suppressed oxidative stress and improved cell viability.
The present findings show for the first time that Hgb is expressed in cervical carcinoma cells and may act as an antioxidant, attenuating oxidative stress-induced damage in cervical cancer cells. These data provide a significant impact not only in globin biology but also in understanding of cervical cancer pathogenesis associated with oxidative stress.
Since the 1980s, epidemics of enterovirus 71 (EV71) and other enteroviruses have occurred in Asian countries and regions, causing a wide range of human diseases. No effective therapy is available for the treatment of these infections. Internal ribosome entry sites (IRESs) are indispensable for the initiation of translation in enteroviruses. Several cellular factors, as well as the ribosome, are recruited to the conserved IRES during this process. Quinacrine intercalates into the RNA architecture and inhibits RNA transcription and protein synthesis, and a recent study showed that quinacrine inhibited encephalomyocarditis virus and poliovirus IRES-mediated translation in vitro without disrupting internal cellular IRES. Here, we report that quinacrine was highly active against EV71, protecting cells from EV71 infection. Replication of viral RNA, expression of viral capsid protein, and production of virus were all strongly inhibited by quinacrine. Interaction of the polypyrimidine tract-binding protein (PTB) with the conserved IRES was prevented by quinacrine. Coxsackieviruses and echovirus were also inhibited by quinacrine in cultured cells. These results indicate that quinacrine may serve as a potential protective agent for use in the treatment of patients with chronic enterovirus infection.
The interplay between tumor cells and their microenvironment plays a pivotal role in tumor development and progression. Although a growing body of evidence has established the importance of the tumor microenvironment, an understanding of the crosstalk between its components and cancer cells remains elusive. The pathways triggered by microenvironmental factors could modulate cancer-related gene transcription, also affecting small noncoding RNAs, microRNAs, which have emerged as key posttranscriptional regulators of gene expression, directly involved in human cancers. Although microRNAs regulate most biological mechanisms, their role in the tumor microenvironment has only recently become the focus of intense research. In this paper, we focus on the intertwined connection between the tumor microenvironment and aberrant expression of microRNAs involved in carcinogenesis. We also discuss the emerging roles of microRNAs in the tumor microenvironment as it relates to cancer progression. We conclude that microRNAs are critical for our understanding of the development of cancer, and that targeting microRNA signaling pathways in the microenvironment as well as in tumor cells opens new therapeutic avenues to the global control of cancer.
The spatiotemporal expression patterns of microRNAs (miRNAs) are important to the verification of their predicted function. RT-qPCR is the accepted technique for the quantification of miRNA expression; however, stem-loop RT-PCR and poly(T)-adapter assay, the two most frequently used methods, are not very convenient in practice and have poor specificity, respectively.
We have developed an optimal approach that integrates these two methods and allows specific and rapid detection of tiny amounts of sample RNA and reduces costs relative to other techniques. miRNAs of the same sample are polyuridylated and reverse transcribed into cDNAs using a universal poly(A)-stem-loop RT primer and then used as templates for SYBR® Green real-time PCR. The technique has a dynamic range of eight orders of magnitude with a sensitivity of up to 0.2 fM miRNA or as little as 10 pg of total RNA. Virtually no cross-reaction is observed among the closely-related miRNA family members and with miRNAs that have only a single nucleotide difference in this highly specific assay. The spatial constraint of the stem-loop structure of the modified RT primer allowed detection of miRNAs directly from cell lysates without laborious total RNA isolation, and the poly(U) tail made it possible to use multiplex RT reactions of mRNA and miRNAs in the same run.
The cost-effective RT-qPCR of miRNAs with poly(A)-stem-loop RT primer is simple to perform and highly specific, which is especially important for samples that are precious and/or difficult to obtain.
Indehiscent sporangia are reported for only a few of derived leptosporangiate ferns. Their evolution has been likely caused by conditions in which promotion of self-fertilization is an evolutionary advantageous strategy such as the colonization of isolated regions and responds to stressful habitat conditions. The Lepisorus clathratus complex provides the opportunity to test this hypothesis because these derived ferns include specimens with regular dehiscent and irregular indehiscent sporangia. The latter occurs preferably in well-defined regions in the Himalaya. Previous studies have shown evidence for multiple origins of indehiscent sporangia and the persistence of populations with indehiscent sporangia at extreme altitudinal ranges of the Qinghai-Tibetan Plateau (QTP).
Independent phylogenetic relationships reconstructed using DNA sequences of the uniparentally inherited chloroplast genome and two low-copy nuclear genes confirmed the hypothesis of multiple origins of indehiscent sporangia and the restriction of particular haplotypes to indehiscent sporangia populations in the Lhasa and Nyingchi regions of the QTP. In contrast, the Hengduan Mountains were characterized by high haplotype diversity and the occurrence of accessions with and without indehiscent sporangia. Evidence was found for polyploidy and reticulate evolution in this complex. The putative case of chloroplast capture in the Nyingchi populations provided further evidence for the promotion of isolated but persistent populations by indehiscent sporangia.
The presented results confirmed the hypothesis that indehiscent sporangia promote the establishment of persistent population in different regions of the QTP. These results are consistent with the expectations of reproductive reassurance by promotion of self-fertilization that played a critical role in the assembly of populations in isolated locations and/or extreme habitats.
Chloroplast capture; Chloroplast DNA; Himalaya; Incongruent phylogenetic hypotheses; Lepisorus clathratus; Phylogenetic analyses; Reticulate evolution; Self-fertilization; Low-copy nuclear genes; PgiC; LFY
Enterovirus 71 (EV71), a member of the Picornaviridae family, is found in Asian countries where it causes a wide range of human diseases. No effective therapy is available for the treatment of these infections. Picornaviruses undergo RNA replication in association with membranes of infected cells. COPI and COPII have been shown to be involved in the formation of picornavirus-induced vesicles. Replication of several picornaviruses, including poliovirus and Echovirus 11 (EV11), is dependent on COPI or COPII. Here, we report that COPI, but not COPII, is required for EV71 replication. Replication of EV71 was inhibited by brefeldin A and golgicide A, inhibitors of COPI activity. Furthermore, we found EV71 2C protein interacted with COPI subunits by co-immunoprecipitation and GST pull-down assay, indicating that COPI coatomer might be directed to the viral replication complex through viral 2C protein. Additionally, because the pathway is conserved among different species of enteroviruses, it may represent a novel target for antiviral therapies.
Viral infectious diseases represent a major threat to public health and are among the greatest disease burdens worldwide. Rapid and accurate identification of viral agents is crucial for both outbreak control and estimating regional disease burdens. Recently developed metagenomic methods have proven to be powerful tools for simultaneous pathogen detection. Here, we performed a systematic study of the capability of the short-read-based metagenomic approach in the molecular detection of viral pathogens in nasopharyngeal aspirate samples from patients with acute lower respiratory tract infections (n = 16). Using the high-throughput capacity of ultradeep sequencing and a dedicated data interpretation method, we successfully identified seven species of known respiratory viral agents from 15 samples, a result that was consistent with results of conventional PCR assays. We also detected a coinfected case that was missed by regular PCR testing. Using the metagenomic data, 11 draft genomes of the abundantly detected viruses in the samples were reconstructed with 21.84% to 98.53% coverage. Our results show the power of the short-read-based metagenomic approach for accurate and parallel screening of viral pathogens. Although there are some inherent difficulties in applying this approach to clinical samples, including a lack of controls, limited specimen quantity, and high contamination rate, our work will facilitate further application of this unprecedented high-throughput method to clinical samples.
EV71 is the primary pathogenic cause of hand-foot-mouth disease (HFMD), but an effective antiviral drug currently is unavailable. Rupintrivir, an inhibitor against human rhinovirus (HRV), has potent antiviral activities against EV71. We determined the high-resolution crystal structures of the EV71 3Cpro/rupintrivir complex, showing that although rupintrivir interacts with EV71 3Cpro similarly to HRV 3Cpro, the C terminus of the inhibitor cannot accommodate the leaving-group pockets of EV71 3Cpro. Our structures reveal that EV71 3Cpro possesses a surface-recessive S2′ pocket that is not present in HRV 3Cpro that contributes to the additional substrate binding affinity. Combined with mutagenic studies, we demonstrated that catalytic Glu71 is irreplaceable for maintaining the overall architecture of the active site and, most importantly, the productive conformation of catalytic His40. We discovered the role of a previously uncharacterized residue, Arg39 of EV71 3Cpro, that can neutralize the negative charge of Glu71, which may subsequently assist deprotonation of His40 during proteolysis.
New therapeutic tools and molecular targets are needed for treatment of Japanese encephalitis virus (JEV) infections. JEV requires an α-1 translational frameshift to synthesize the NS1' protein required for viral neuroinvasiveness. Several flavonoids have been shown to possess antiviral activity in vitro against a wide spectrum of viruses. To date, the antiviral activities of flavonol kaempferol (Kae) and isoflavonoid daidzin (Dai) against JEV have not been described.
The 50% cytotoxic concentration (CC50) and 50% effective concentration (EC50) against JEV were investigated in BHK21 cells by MTS reduction. Activity against viral genomic RNA and proteins was measured by real-time RT-PCR and western blotting. The frameshift site RNA-binding characterization was also determined by electrospray ionization mass spectrometry, isothermal titration calorimetry and autodocking analysis. EC50 values of Kae and Dai were 12.6 and 25.9 µM against JEV in cells pretreated before infection, whereas in cells infected before treatment, EC50 was 21.5 and 40.4 µM, respectively. Kae exhibited more potent activity against JEV and RNA binding in cells following internalization through direct inhibition of viral replication and protein expression, indicating that its antiviral activity was principally due to direct virucidal effects. The JEV frameshift site RNA (fsRNA) was selected as a target for assaying Kae and Dai. ITC of fsRNA revealed an apparent Kb value for Kae that was nine fold stronger than that for Dai. This binding was confirmed and localized to the RNA using ESI-MS and autodock analysis. Kae could form non-covalent complexes with fsRNA more easily than Dai could.
Kae demonstrates more potent antiviral activity against JEV than does Dai. The mode of action of Kae as an anti-JEV agent seems to be related to its ability to inactivate virus by binding with JEV fsRNA.
Japanese encephalitis virus (JEV), a neurotropic mosquito-borne flavivirus, causes acute viral encephalitis and neurologic disease with a high fatality rate in humans and a range of animals. Small interfering RNA (siRNA) is a powerful antiviral agent able to inhibit JEV replication. However, the high rate of genetic variability between JEV strains (of four confirmed genotypes, genotypes I, II, III and IV) hampers the broad-spectrum application of siRNAs, and mutations within the targeted sequences could facilitate JEV escape from RNA interference (RNAi)-mediated antiviral therapy. To improve the broad-spectrum application of siRNAs and prevent the generation of escape mutants, multiple siRNAs targeting conserved viral sequences need to be combined. In this study, using a siRNA expression vector based on the miR-155 backbone and promoted by RNA polymerase II, we initially identified nine siRNAs targeting highly conserved regions of seven JEV genes among strains of the four genotypes of JEV to effectively block the replication of the JEV vaccine strain SA14-14-2. Then, we constructed single microRNA-like polycistrons to simultaneously express these effective siRNAs under a single RNA polymerase II promoter. Finally, these single siRNAs or multiple siRNAs from the microRNA-like polycistrons showed effective anti-virus activity in genotype I and genotype III JEV wild type strains, which are the predominant genotypes of JEV in mainland China. The anti-JEV effect of these microRNA-like polycistrons was also predicted in other genotypes of JEV (genotypes II and IV), The inhibitory efficacy indicated that siRNAs×9 could theoretically inhibit the replication of JEV genotypes II and IV.
The DNA damage response (DDR) encompasses multi-step processes by which cells evolve to sense DNA damage, transduce the signal and initiate the repair of damaged DNA. Ataxia Telangiectasia Mutated (ATM) Kinase, which functions as the primary sensor and transducer of DNA damage signal, has been demonstrated to play an important role in the DDR and cancer prevention. Hence, understanding the molecular mechanisms underlying the regulation of ATM has received much attention. Here, we found that miR-18a was upregulated in both cell lines and patients' tissue samples of breast cancer. Furthermore, we demonstrated that ectopically expressing miR-18a downregulated ATM expression by directly targeting the ATM-3′-UTR and abrogated the IR-induced cell cycle arrest. Similar to the effect of ATM siRNA, overexpressing miR-18a in breast cancer cells reduced the DNA damage repair ability and the efficiency of homologous recombination-based DNA repair (HRR) and sensitized cells to γ-irradiation (IR) treatment. However, inhibition of miR-18a led to augmentation of DNA damage repair, increase of HRR efficiency and reduced cellular radiosensitivity. Moreover, we showed that the phorsphorylation level and nuclear foci formation of H2AX and 53BP1, the downstream substrates of ATM kinase, were significantly deceased in miR-18a overexpressing cells. Taken together, our results uncover a new regulatory mechanism of ATM expression and suggest that miR-18a might be a novel therapeutic target.
Nuclear factor κB (NF-κB)-mediated pathways have been widely implicated in cell survival, development and tumor progression. Although the molecular events of determining NF-κB translocation from cytoplasm to nucleus have been extensively documented, the regulatory mechanisms of NF-κB activity inside the nucleus are still poorly understood. Being a special member of macro domain proteins, LRP16 was previously identified as a coactivator of both estrogen receptor and androgen receptor, and as an interactor of NF-κB coactivator UXT. Here, we investigated the regulatory role of LRP16 on NF-κB activation.
GST pull-down and coimmunoprecipitation (CoIP) assays assessed protein-protein interactions. The functional activity of NF-κB was assessed by luciferase assays, changes in expression of its target genes, and its DNA binding ability. Annexin V staining and flow cytometry analysis were used to evaluate cell apoptosis. Immunohistochemical staining of LRP16 and enzyme-linked immunosorbent assay-based evaluation of active NF-κB were performed on primary human gastric carcinoma samples.
We demonstrate that LRP16 integrates into NF-κB transcriptional complex through associating with its p65 component. RNA interference knockdown of the endogenous LRP16 in cells leads to impaired NF-κB activity and significantly attenuated NF-κB-dependent gene expression. Mechanistic analysis revealed that knockdown of LRP16 did not affect tumor necrosis factor α (TNF-α)-induced nuclear translocation of NF-κB, but blunted the formation or stabilization of functional NF-κB/p300/CREB-binding protein transcription complex in the nucleus. In addition, knockdown of LRP16 also sensitizes cells to apoptosis induced by TNF-α. Finally, a positive link between LRP16 expression intensity in nuclei of tumor cells and NF-κB activity was preliminarily established in human gastric carcinoma specimens.
Our findings not only indicate that LRP16 is a crucial regulator for NF-κB activation inside the nucleus, but also suggest that LRP16 may be an important contributor to the aberrant activation of NF-κB in tumors.
Overexpression of sphingosine kinase-1 (SPHK1) has been demonstrated to be associated with the development and progression in various types of human cancers. The current study was to characterize the expression of SPHK1 in salivary gland carcinomas (SGC) and to investigate the association between SPHK1 expression and progression of SGC.
The expression of SPHK1 was examined in 2 normal salivary gland tissues, 8 SGC tissues of various clinical stages, and 5 pairs of primary SGC and adjacent salivary gland tissues from the same patient, using real-time PCR and western blot analysis. Furthermore, the SPHK1 protein expression was analyzed in 159 clinicopathologically characterized SGC cases by immunohistochemistry. Statistical analyses were performed to determine the prognostic and diagnostic associations.
SPHK1 expression was found to be markedly upregulated in SGC tissues than that in the normal salivary gland tissues and paired adjacent salivary gland tissues, at both mRNA and protein levels. Statistical analysis revealed a significant correlation of SPHK1 expression with the clinical stage (P = 0.005), T classification (P = 0.017), N classification (P = 0.009), M classification (P = 0.002), and pathological differentiation (P = 0.013). Patients with higher SPHK1 expression had shorter overall survival time, whereas patients with lower SPHK1 expression had better survival. Importantly, patients in the group without adjuvant therapy who exhibited high SPHK1 expression had significantly lower overall survival rates compared with those with low SPHK1 expression. Moreover, multivariate analysis suggested that SPHK1 expression might be an independent prognostic indicator for the survival of SGC patients.
Our results suggest that SPHK1 expression is associated with SGC progression, and might represent as a novel and valuable predictor for adjuvant therapy to SGC patients.
Oncogenesis in breast cancer is often associated with excess estrogen receptor α(ERα) activation and overexpression of its coactivators. LRP16 is both an ERα target gene and an ERα coactivator, and plays a crucial role in ERα activation and proliferation of MCF-7 breast cancer cells. However, the regulation of the functional availability of this coactivator protein is not yet clear.
Yeast two-hybrid screening, GST pulldown and coimmunoprecipitation (CoIP) identified the cytoplasmic intermediate filament protein keratin 18 (K18) as a novel LRP16-interacting protein. Fluorescence analysis revealed that GFP-tagged LRP16 was primarily localized in the nuclei of mock-transfected MCF-7 cells but was predominantly present in the cytoplasm of K18-transfected cells. Immunoblotting analysis demonstrated that the amount of cytoplasmic LRP16 was markedly increased in cells overexpressing K18 whereas nuclear levels were depressed. Conversely, knockdown of endogenous K18 expression in MCF-7 cells significantly decreased the cytoplasmic levels of LRP16 and increased levels in the nucleus. CoIP failed to detect any interaction between K18 and ERα, but ectopic expression of K18 in MCF-7 cells significantly blunted the association of LRP16 with ERα, attenuated ERα-activated reporter gene activity, and decreased estrogen-stimulated target gene expression by inhibiting ERα recruitment to DNA. Furthermore, BrdU incorporation assays revealed that K18 overexpression blunted the estrogen-stimulated increase of S-phase entry of MCF-7 cells. By contrast, knockdown of K18 in MCF-7 cells significantly increased ERα-mediated signaling and promoted cell cycle progression.
K18 can effectively associate with and sequester LRP16 in the cytoplasm, thus attenuating the final output of ERα-mediated signaling and estrogen-stimulated cell cycle progression of MCF-7 breast cancer cells. Loss of K18 increases the functional availability of LRP16 to ERα and promotes the proliferation of ERα-positive breast tumor cells. K18 plays an important functional role in regulating the ERα signaling pathway.
Inhibitor of differentiation 2 (Id2) is a natural inhibitor of the basic helix–loop–helix transcription factors. Although Id2 is well known to prevent differentiation and promote cell-cycle progression and tumorigenesis, the molecular events that regulate Id2 activity remain to be investigated. Here, we identified that Four-and-a-half LIM-only protein 2 (FHL2) is a novel functional repressor of Id2. Moreover, we demonstrated that FHL2 can directly interact with all members of the Id family (Id1–4) via an N-terminal loop–helix structure conserved in Id proteins. FHL2 antagonizes the inhibitory effect of Id proteins on basic helix–loop–helix protein E47-mediated transcription, which was abrogated by the deletion mutation of Ids that disrupted their interaction with FHL2. We also showed a competitive nature between FHL2 and E47 for binding Id2, whereby FHL2 prevents the formation of the Id2–E47 heterodimer, thus releasing E47 to DNA and restoring its transcriptional activity. FHL2 expression was remarkably up-regulated during retinoic acid-induced differentiation of neuroblastoma cells, during which the expression of Id2 was opposite to that. Ectopic FHL2 expression in neuroblastoma cells markedly reduces the transcriptional and cell-cycle promoting functions of Id2. Altogether, these results indicate that FHL2 is an important repressor of the oncogenic activity of Id2 in neuroblastoma cells.
Inhibitor of differentiation 2 (Id2) is a critical factor for cell proliferation and differentiation in normal vertebrate development. Most of the biological function of Id2 has been ascribed to its helix-loop-helix motif. Overexpression of Id2 is frequently observed in various human tumors, but its role for invasion potential in tumor cells is dispute. We aimed to reveal the role of Id2 in invasion potential in poorly invasive and estrogen receptor α (ERα)-positive MCF-7 and SKOV-3 cancer cells.
MCF-7 and SKOV-3 cells were stably transfected with the wild-type, degradation-resistant full-length or helix-loop-helix (HLH)-deleted Id2, respectively. Protein levels of Id2 and its mutants and E-cadherin were determined by western blot analysis and mRNA levels of Id2 and its mutants were determined by RT-PCR. The effects of Id2 and its mutants on cell proliferation were determined by [3H]-thymidine incorporation assay and the 3- [4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) dye method. The in vitro invasion potential of cells was evaluated by Transwell assay. Cell motility was assessed by scratch wound assay. The promoter activity of E-cadherin was determined by cotransfection and luciferase assays.
Ectopic transfection of the wild-type Id2 markedly increased the protein and mRNA expression of Id2 in MCF-7 and SKOV-3 cells; the protein level but not mRNA level was further increased by transfection with the degradation-resistant Id2 form. The ectopic expression of Id2 or its mutants did not alter proliferation of either MCF-7 or SKOV-3 cells. Transfection of the wild-type Id2 significantly induced the invasion potential and migratory capacity of cells, which was further augmented by transfection with the degradation-resistant full-length or HLH-deleted Id2. E-cadherin protein expression and transactivation of the proximal E-cadherin promoter were markedly suppressed by the degradation-resistant full-length or HLH-deleted Id2 but not wild-type Id2. Ectopic expression of E-cadherin in MCF-7 and SKOV-3 cells only partially blunted the invasion potential induced by the degradation-resistant HLH-deleted Id2.
Overexpression of Id2 in ERα-positive epithelial tumor cells indeed increases the cells' invasive potential through a novel mechanism independent of dimerization to basic helix-loop-helix factors. E-cadherin contributes only in part to Id2-induced cell invasion when Id2 is accumulated to a higher level in some specific cell types.
Dynamic spectrum access is a must-have ingredient for future sensors that are ideally cognitive. The goal of this paper is a tutorial treatment of wideband cognitive radio and radar—a convergence of (1) algorithms survey, (2) hardware platforms survey, (3) challenges for multi-function (radar/communications) multi-GHz front end, (4) compressed sensing for multi-GHz waveforms—revolutionary A/D, (5) machine learning for cognitive radio/radar, (6) quickest detection, and (7) overlay/underlay cognitive radio waveforms. One focus of this paper is to address the multi-GHz front end, which is the challenge for the next-generation cognitive sensors. The unifying theme of this paper is to spell out the convergence for cognitive radio, radar, and anti-jamming. Moore’s law drives the system functions into digital parts. From a system viewpoint, this paper gives the first comprehensive treatment for the functions and the challenges of this multi-function (wideband) system. This paper brings together the inter-disciplinary knowledge.
cognitive radio; testbed; cognitive radar