Substitutions of stem-loop subdomains in internal ribosome entry site of Senecavirus A:Impacts on rescue of sequence-modifying viruses

Senecavirus A(SVA)has a positive-sense,single-stranded RNA genome.Its 5´untranslated region harbors an internal ribosome entry site(IRES),comprising 10 larger or smaller stem-loop structures(including a pseudoknot)that have been demonstrated to be well conserved.However,it is still unclear whether each stem-loop subdomain,such as a single stem or loop,is also highly conserved.To clarify this issue in the present study,a set of 29 SVA cDNA clones were constructed by site-directed mutagenesis(SDM)on the IRES.The SDM-modified scenarios included:(1)stem-formed complementary sequences exchanging with each other;(2)loop transversion;(3)loop transition;and(4)point mutations.All cDNA clones were separately transfected into cells for rescuing viable viruses,whereas only four SVAs of interest could be recovered,and were genetically stable during 20 passages.One progeny grew significantly slower than the other three did.The dual-luciferase reporter assay showed that none of the SDM-modified IRESes significantly inhibited the IRES activity.Our previous study indicated that a single motif from any of the ten stem structures,if completely mutated,would cause the failure of virus recovery.Interestingly,our present study revealed three stem structures,whose individual complementary sequences could exchange with each other to rescue sequence-modifying SVAs.Moreover,one apical loop was demonstrated to have the ability to tolerate its own full-length transition,also having no impact on the recovery of sequence-modifying SVA.The present study suggested that not every stem-loop structure was strictly conserved in its conformation,while the full-length IRES itself was well conserved.This provides a new research direction on interaction between the IRES and many factors.

Detection of Drug Resistance in Escherichia coli Isolated from Animals and ERIC Analysis of Multidrug-resistant Strains

The drug resistance of Escherichia coli from animals was detected in Shandong Province in 2016,the gene mapping of Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR) of multidrug-resistant isolates were analyzed,and then the relationship between ERIC-PCR genotyping and drug resistance of highly-resistant E.coli with multidrug resistance was discussed.A total of 110 E.coli isolates were separated and identified from diseased swine and avian,and their resistance to 10 kinds of antimicrobials was determined by the agar dilution method.Twenty highly-resistant isolates with multidrug resistance were selected to carry on the ERIC-PCR,followed by cluster genetic analysis according to the DNA fingerprints.The swine-sourced E.coli isolates possessed serious resistance against several kinds of antimicrobial agents,for example,all isolates were tolerant to florfenicol,doxycycline and ampicillin (100%),but had a relative lower resistance rate to cefotaxime sodium (57.14%).The same situation was observed in the poultry-sourced E.coli isolates,which had a resistance rate of 95.51% against florfenicol,while the lowest rate of 61.80% appeared on ciprofloxacin.Analysis of ERTIC showed dispersive fingerprint patterns of highly-resistant and multidrug- resistant isolates which represented a multiple clone resources,and there were certain correlation between the B genotype and the drug-resistant characteristics.It indicated that the animal-sourced E.coli isolates had a high level of drug resistance and were multidrug-resistant,which meant there was severe antibiotic resistance against not only different kinds of antibiotics but also different drugs of the same kind.The highly-resistant E.coli isolates with multidrug resistance had no apparent species preference,while their spread and pervasion posed a potential threat to the development of animal husbandry and the public health security.

GreenPhos,a universal method for in-depth measurement of plant phosphoproteomes with high quantitative reproducibility

Protein phosphorylation regulates a variety of important cellular and physiological processes in plants.In-depth profiling of plant phosphoproteomes has been more technically challenging than that of animal phosphoproteomes.This is largely due to the need to improve protein extraction efficiency from plant cells,which have a dense cell wall,and to minimize sample loss resulting from the stringent sample clean-up steps required for the removal of a large amount of biomolecules interfering with phosphopeptide purification and mass spectrometry analysis.To this end,we developed a method with a streamlined workflow for highly efficient purification of phosphopeptides from tissues of various green organisms including Arabidopsis,rice,tomato,and Chlamydomonas reinhardtii,enabling in-depth identification with high quantitative reproducibility of about 11000 phosphosites,the greatest depth achieved so far with single liquid chromatography-mass spectrometry(LC-MS)runs operated in a data-dependent acquisition(DDA)mode.The mainstay features of the method are the minimal sample loss achieved through elimination of sample clean-up before protease digestion and of desalting before phosphopeptide enrichment and hence the dramatic increases of time-and cost-effectiveness.The method,named GreenPhos,combined with single-shot LC-MS,enabled in-depth quantitative identification of Arabidopsis phosphoproteins,including differentially phosphorylated spliceosomal proteins,at multiple time points during salt stress and a number of kinase substrate motifs.GreenPhos is expected to serve as a universal method for purification of plant phosphopeptides,which,if samples are further fractionated and analyzed by multiple LC-MS runs,could enable measurement of plant phosphoproteomes with an unprecedented depth using a given mass spectrometry technology.

Promotional effect of Cu additive for the selective catalytic oxidation of n-butylamine over CeZrO_(x)catalyst

The catalytic elimination of nitrogen-containing volatile organic compounds(NVOCs)still encounters bottlenecks in NO_(x)formation and low N_(2)selectivity.Here,a series of Cu-promoted Ce-Zr mixed oxide catalysts were synthesized using a simple precipitation approach,and n-butylamine was adopted as the probe pollutant to evaluate their catalytic performance.The Ce Cu_(10%)ZrO_(x)catalyst exhibited the best catalytic activity,with 100%n-butylamine conversion and 90%N_(2)selectivity at 250℃.Concurrently,this sample also displayed good water resistance.A detailed characterization of the catalyst was performed through a series of experimental studies and theoretical calculations.The addition of Cu increased the redox property and promoted the production of oxygen vacancies,all of which were favorable for the greatest nbutylamine selective catalytic oxidation performance.The changes of oxygen vacancies over Ce Cu_(10%)ZrO_(x)in reaction process were studied by in situ Raman spectra.Moreover,in situ diffuse reflectance infrared Fourier transform spectra(DRIFTs)and theoretical calculations were employed to explore the reaction mechanism of n-butylamine selective oxidation.The high activity and selectivity of this catalyst confirm the practical feasibility of the selective oxidation of n-butylamine to CO_(2)and N_(2),and the exploration of the reaction mechanism provides new insights into the further design of catalysts.