Activation of 2-5A-dependent ribonuclease by 5'-phosphorylated, 2',5'-linked oligoadenylates, known as 2-5A, is one pathway of interferon action. Unaided uptake into HeLa cells of 2-5A linked to an antisense oligonucleotide resulted in the selective ablation of messenger RNA for the double-stranded RNA (dsRNA)-dependent protein kinase PKR. Similarly, purified, recombinant human 2-5A-dependent ribonuclease was induced to selectively cleave PKR messenger RNA. Cells depleted of PKR activity were unresponsive to activation of nuclear factor-kappa B (NF-kappa B) by the dsRNA poly(I):poly(C), which provides direct evidence that PKR is a transducer for the dsRNA signaling of NF-kappa B.

Composite nucleic acids, known as 2-5A antisense chimeras, cause the 2-5A-dependent ribonuclease (RNase L) to catalyze the specific cleavage of RNA in cell free systems and in intact cells. Such 2-5A antisense chimeras are 5'-monophosphorylated, 2,'5'-linked oligoadenylates covalently attached to antisense 3',5'-oligodeoxyribonucleotides by means of a linker containing two residues of 1,4-butanediol phosphate. Here we report a fully automated synthesis of 2-5A antisense chimeras on a solid support using phosphoramidite methodology with specific coupling time modifications and their subsequent purification by reverse-phase ion-pair and anion exchange HPLC. Purified 2-5A antisense chimeras were characterized by [1H]NMR and [31P]NMR, MALDIMS, and capillary gel electrophoresis. The synthetic 2',5'-linked oligoadenylate showed no phosphodiester isomerization to 3',5' during or after synthesis. In addition, we have developed facile methodologies to characterize the chimeras using digestion with various hydrolytic enzymes including snake venom phosphodiesterase I and nuclease P1. Finally, Maxam-Gilbert chemical sequencing protocols have been developed to confirm the entire sequence of these chimeric oligonucleotides.

2-5A antisense (2-5A-AS) molecules are chimeric oligonucleotides that cause 2-5A-dependent RNase (RNase L) to catalyze the selective cleavage of RNA in human cells. These composite nucleic acids consist of a 5'-monophosphorylated, 2',5'-linked oligoadenylate known as 2-5A (an activator of RNase L) covalently attached to antisense 3',5'-oligodeoxyribonucleotides. Here, we characterize the targeted cleavage of the double-stranded RNA-dependent protein kinase (PKR) mRNA by purified, recombinant human RNase L. A 2-5A-AS chimera, which contains complementary sequence to PKR mRNA, and unmodified 2-5A, which causes general RNA decay, were about 20- and 40-fold more active, respectively, than 2-5A-AS chimeras in which the DNA domains are not complementary to sequences in PKR mRNA. Directed cleavage was efficient because each 2-5A-AS chimera targeted many RNA molecules. Moreover, RNase L caused the catalytic cleavage of the RNA target (kcat of approximately 7 s-1). The precise sites of PKR mRNA cleavage caused by 2-5A-AS were mapped, using a primer extension assay, to phosphodiester bonds adjacent to the 3' terminus of the chimera binding site (5' on the RNA target) as well as within the chimera's oligonucleotide binding site itself. The selectivity of this approach is shown to be provided by the antisense arm of the chimera, which places the RNA target in close proximity to the RNase.

ObjectiveTo investigate the acute effect of air pollutants on ischaemic stroke (IS) and IS-related death.SettingFive urban districts in Changzhou, China, between 9 January 2015 and 31 December 2016.ParticipantsA total of 32 840 IS cases and 4028 IS deaths were enrolled.Main outcome measuresA time-series design, generalised additive model and multivariable regression model were used to examine the percentage change (95% CI) in daily IS counts and deaths with an IQR increase in air pollutant levels for different single or multiple lag days in single-pollutant and two-pollutant models.ResultsDaily IS counts increased 0.208% (95% CI 0.036% to 0.381%) with an IQR increment in the levels of nitrogen dioxide (NO2). The estimated risk of NO2 was more robust in males and in the cold season. For daily IS counts, the estimated effects of NO2 and sulfur dioxide (SO2) were more significant when adjusted for particulate matter with aerodynamic diameters <2.5 µm (PM2.5) and PM10. An IQR increment in the concentration of PM10, SO2 and NO2 significantly increased IS deaths with 6 days of cumulative effects (0.268%, 95% CI 0.007% to 1.528%; 0.34%, 0.088% to 0.592%; and 0.263%, 0.004% to 0.522%, respectively). Young individuals (<65 years old) had a higher IS mortality risk for PM2.5, PM10, NO2 and CO. For IS death, the effect estimates of SO2 in the elderly, females and the cold season were more pronounced; statistical significance was also identified for SO2 when adjusted for carbon monoxide (CO).ConclusionsThis study suggested that short-term exposure to ambient NO2 was associated with increased IS risk. In addition, SO2 was associated with increased IS onset and death.

Treatment of human cells with 2 ,5 oligoadenylate covalently linked to antisense (2-5A-antisense) results in the selective cleavage of targeted RNA species by 2-5A-dependent RNase L. Here we show that 2-5A-antisense containing stabilizing modifications at both termini are effective in suppressing the replication of respiratory syncytial virus (RSV) in human tracheal epithelial cells. The affinity of 2-5A-antisense for different regions in the RSV M2 and L mRNAs was predicted from a computer-generated model of the RNA secondary structure. The most potent 2-5A-antisense molecule caused a highly effective, dose-dependent suppression of RSV yields when added to previously infected cells. In contrast, control oligonucleotides, including an inactive dimeric form of 2-5A linked to antisense, 2-5A linked to a randomized sequence of nucleotides, and antisense molecules lacking 2-5A, had minimal effects on virus replication. The specificity of this approach was shown by reverse transcriptase-coupled PCR analysis of RSV M2, P, and N mRNA and of cellular glyceraldehyde-3-phosphate dehydrogenase mRNA. The RSV M2 mRNA amounts were depleted after treating RSV-infected cells with 2-5A-antisense targeted to this mRNA, whereas the amounts of the other RNA species were unchanged. These studies demonstrate that 2 ,5 oligoadenylate covalently linked to antisense (2-5A-antisense) can effectively suppress RSV replication by directing the cellular RNase L to selectively degrade an essential viral mRNA.Respiratory syncytial virus (RSV), a nonsegmented, negativestrand RNA virus in the pneumovirus genus of Paramyxoviridae, is a major cause of lower respiratory disease, resulting in 90,000 hospitalizations and 4500 deaths per year in infants and young children in the United States (1). Of growing concern are outbreaks of RSV within institutionalized elderly (2) and immunodeficient (1) adults. There is only one approved anti-RSV compound, ribavirin, which reduces virus shedding in infected children, but has no effect on mortality or duration of hospitalization (3). In the search for alternative antiviral strategies, we have investigated the potential of novel chimeric antisense molecules, 2-5A-antisense, in the control of RSV infections.Zamecnik and Stephenson were the first to report an antiviral effect of antisense oligonucleotides by showing that replication of the retrovirus Rous sarcoma virus could be inhibited by the addition of oligonucleotides complementary in sequence to reiterated terminal sequences of the viral 35S RNA (4). Subsequently, antisense oligonucleotides have been used against a wide range of different types of viruses, including the negative-strand RNA viruses such as rabies virus (5) and influenza virus (6), positive-strand RNA viruses such as dengue virus (7), DNA viruses including hepatitis B (8) and herpes simplex virus type 1 (9), and the retrovirus HIV-1 (10, 11). Despite encouraging results, significant challenges remain in the development of antisense oligonucleotides as antiviral agents. For instance, ...

Our study revealed a novel mechanism in the CaA-attenuated angiogenic ability in HCC cells possiblyviareducing the JNK-1-mediated HIF-1α stabilization and inducing the ubiquitination-mediated HIF1α degradation.

BackgroundSexual transmission of HIV among men who have sex with men (MSM) increased markedly in China during the past decade. HIV incidence is a critical indicator in HIV surveillance and we use a HIV-1 BED-capture-enzyme immunoassay (BED-CEIA) to examine the incidence among MSM in Beijing from 2008 to 2016. Risk factors related to recent HIV infection were also assessed.MethodsConsecutive cross-sectional surveys on MSM were conducted yearly from 2008 through 2016. Demographic and behaviors data were collected. HIV status was determined and HIV positive specimens were tested for recent infection using BED-CEIA. Specimens with ODn values≤0.8 were considered recently infected, HIV incidence rates and prevalence were then calculated. Risk factors associated with recent HIV infection were assessed by univariate and multivariable logistic regression.ResultsFrom 2008 to 2016, the numbers of eligible participants in the nine consecutive years ranged from 472 to 616. All the 261 eligible HIV-positive specimens were subjected to recent HIV infection testing. HIV prevalence ranged from 5.0% (3.3%-6.8%) to 10.2% (7.8%-12.7%), and incidence ranged from 1.57% (0.19%-2.95%) to 6.63% (3.65%-9.61%). MSM who never or sometimes used condoms during anal sex with men in the past 6 months (aOR = 1.515, 95%CI: 1.016–2.257, p = 0.041), or having syphilis infection (aOR = 1.561, 95%CI: 0.946–2.575, p = 0.081) were more likely to be recently infected with HIV. Being a Beijing resident (aOR = 0.409, 95%CI: 0.212–0.790, p = 0.008), or having only one male anal sex partner in the past 6 months (aOR = 0.467, 95%CI: 0.220–0.994, p = 0.048) were associated with a lower risk for recent HIV infection.ConclusionsThe HIV incidence fluctuated among MSM in Beijing. Unprotected anal sex, having multiple sex partners, being a non-registered Beijing resident and having a syphilis infection play important roles in the recent HIV infection. Effective intervention measures for HIV and syphilis control and prevention should be continuously strengthened.

A highly N-phosphonomethylglycine (glyphosate)-resistant Pseudomonas fluorescens G2 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) was mapped to identify potential split sites using a transposon-based linker-scanning procedure. Intein-mediated protein complementation was used to reconstitute glyphosate resistance from the genetically divided G2 EPSPS gene in Escherichia coli strain ER2799 and transgenic tobacco.