Afoxolaner is an isoxazoline compound characterized by a good safety profile and extended effectiveness against fleas and ticks on dogs following a single oral administration. In vitro membrane feeding assay data and in vivo pharmacokinetic studies in dogs established an afoxolaner blood concentration of 0.1-0.2 μg/ml to be effective against both fleas (Ctenocephalides felis) and ticks (Dermacentor variabilis). Pharmacokinetic profiles in dogs following a 2.5mg/kg oral dosage demonstrated uniform and predictable afoxolaner plasma concentrations above threshold levels required for efficacy for more than one month. Dose ranging and a 5-month multi-dose experimental study in dogs, established that the 2.5mg/kg oral dosage was highly effective against fleas and ticks, and produced predictable and reproducible pharmacokinetics following repeated dosing. Mode of action studies showed that afoxolaner blocked native and expressed insect GABA-gated chloride channels with nanomolar potency. Afoxolaner has comparable potency between wild type channels and channels possessing the A302S (resistance-to-dieldrin) mutation. Lack of cyclodiene cross-resistance for afoxolaner was confirmed in comparative Drosophila toxicity studies, and it is concluded that afoxolaner blocked GABA-gated chloride channels via a site distinct from the cyclodienes.

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Canker caused by the Ascomycete Valsa mali is the most destructive disease of apple in Eastern Asia, resulting in yield losses of up to 100%. This necrotrophic fungus induces severe necrosis on apple, eventually leading to the death of the whole tree. Identification of necrosis inducing factors may help to unravel the molecular bases for colonization of apple trees by V. mali. As a first step toward this goal, we identified and characterized the V. mali repertoire of candidate effector proteins (CEPs). In total, 193 secreted proteins with no known function were predicted from genomic data, of which 101 were V. mali-specific. Compared to non-CEPs predicted for the V. mali secretome, CEPs have shorter sequence length and a higher content of cysteine residues. Based on transient over-expression in Nicotiana benthamiana performed for 70 randomly selected CEPs, seven V. mali Effector Proteins (VmEPs) were shown to significantly suppress BAX-induced PCD. Furthermore, targeted deletion of VmEP1 resulted in a significant reduction of virulence. These results suggest that V. mali expresses secreted proteins that can suppress PCD usually associated with effector-triggered immunity (ETI). ETI in turn may play an important role in the V. mali–apple interaction. The ability of V. mali to suppress plant ETI sheds a new light onto the interaction of a necrotrophic fungus with its host plant.

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In this study we further defined the rifampin-binding sites in Escherichia coli RNA polymerase (RNAP) and determined the relationship between rifampin-binding sites and the binding sites of other antibiotics, including two rifamycin derivatives, rifabutin and rifapentine, and streptolydigin and sorangicin A, which are unrelated to rifampin, using a purified in vitro system. We found that there is almost a complete correlation between resistance to rifampin (Rif r ) and reduced rifampin binding to 12 RNAPs purified from different rpoB Rif r mutants and a complete cross-resistance among the different rifamycin derivatives. Most Rif r RNAPs were sensitive to streptolydigin, although some exhibited weak resistance to this antibiotic. However, 5 out of the 12 Rif r RNAPs were partially resistant to sorangicin A, and one was completely cross-resistant to sorangicin A, indicating that the binding site(s) for these two antibiotics overlaps. Both rifampin and sorangicin A inhibited the transition step between transcription initiation and elongation; however, longer abortive initiation products were produced in the presence of the latter, indicating that the binding site for sorangicin A is within the rifampin-binding site. Competition experiments of different antibiotics with 3 H-labeled rifampin for binding to wild-type RNAP further confirmed that the binding sites for rifampin, rifabutin, rifapentine, and sorangicin A are shared, whereas the binding sites for rifampin and streptolydigin are distinct. Because Rif r mutations are highly conserved in eubacteria, our results indicate that this set of Rif r mutant RNAPs can be used to screen for new antibiotics that will inhibit the growth of Rif r pathogenic bacteria.RNA polymerase (RNAP) is the sole enzyme responsible for transcribing RNA from DNA template in eubacteria (3). Because of its essential role in gene expression, RNAP has been a target for antibiotic studies since its discovery in the 1960s. Several antibiotics inhibiting the functions of RNAP have been discovered. Among them, rifampin, a derivative of rifamycin (35), is the most important in clinical use (22,33). Rifampin is part of the standard therapy of tuberculosis (8) which, after AIDS, is the leading cause of death by an infectious agent worldwide (23,32) and is also used in prophylaxis of meningitis and against staphylococcal infections (19,20).Rifampin binds to RNAP with high affinity (K eq ϭ 10 Ϫ9 M at 37°C) (39). The mode of action of rifampin has been studied in most detail using Escherichia coli RNAP as a model system, as the overall structure and function of RNAPs from different eubacteria have been conserved. Rifampin inhibits RNAP's function by blocking the transition from transcription initiation to transcription elongation (24). In the presence of rifampin, RNAP can only synthesize short RNA oligomers, and it was proposed that rifampin exerts a steric hindrance of RNAP translocation along the nascent RNA path. Indeed, cross-linking experiments have indicated that rifampin blocks the channel le...

Summary MicroRNAs play important roles in the regulation of gene expression in plants and animals. However, little information is known about the action mechanism and function of fungal microRNA‐like RNAs (milRNAs). In this study, combining deep sequencing, molecular and histological assays, milRNAs and their targets in the phytopathogenic fungus Valsa mali were isolated and identified. A critical milRNA, Vm‐milR16, was identified to adaptively regulate the expression of virulence genes. Fourteen isolated milRNAs showed high expression abundance. Based on the assessment of a pathogenicity function of these milRNAs, Vm‐milR16 was found to be a critical milRNA in V. mali by regulating sucrose non‐fermenting 1 (VmSNF1), 4,5‐DOPA dioxygenase extradiol (VmDODA), and a hypothetical protein (VmHy1). During V. mali infection, Vm‐milR16 is downregulated, while its targets are upregulated. Overexpression of Vm‐milR16, but not mutated Vm‐milR16, significantly reduces the expression of targets and virulence of V. mali. Furthermore, deletion of VmSNF1, VmDODA and VmHy1 significantly reduce virulence of V. mali. All three targets seem to be essential for oxidative stress response and VmSNF1 is required for expression of pectinase genes during V. mali–host interaction. Our results demonstrate Vm‐milRNAs contributing to the infection of V. mali on apple trees by adaptively regulating virulence genes.

(39 isolates, 12.1%). Overall, S. Typhimurium was the most commonly detected serovar; among the individual species, S. Pullorum was most commonly isolated from chickens, S. Enteritidis was most common in ducks, S. Typhimurium was most common in geese and pigeons, and S. enterica serovar Saintpaul was most common in turkeys. PCR determination of 20 fimbrial genes demonstrated the presence of bcfD, csgA, fimA, stdB, and sthE genes and the absence of staA and stgA genes in these isolates, and other loci were variably distributed, with frequency values ranging from 11.8 to 99.1%. These 323 Salmonella isolates were subdivided into 41 different fimbrial genotypes, and of these isolate, 285 strains (88.2%) had 12 to 14 fimbrial genes. Our findings indicated that the Salmonella isolates from different poultry species were phenotypically and genetically diverse and that some fimbrial genes are more frequently associated with serovars or serogroups.

Aims:  In this study, we examined the biofilm formation of 75 Salmonella enterica serovar Typhimurium (Salm. Typhimurium) human clinical isolates and the effect of subinhibitory concentrations (sub‐MICs) of gentamicin, ciprofloxacin and cefotaxime on biofilm formation and exopolysaccharides (EPS) production. Methods and Results:  Quantification of biofilm formation and EPS production were carried out using a modified microtitre plate assay and spectrophotometric method, respectively. The results indicate that 38 isolates (50·7%), which are predominantly of DT104 phage type, presented as the strong biofilm producers in vitro on plastic surface. When strains with the highest biofilm‐forming capacity were grown in the presence of sub‐MICs of gentamicin and ciprofloxacin, the inhibition of biofilm formation and EPS production was observed. In contrast, cefotaxime at 1/2 MIC (0·039 μg ml−1) was able to significantly induce the production of biofilm as well as EPS in three isolates with nontypable and DT104 phage type, respectively. Conclusions:  These results clearly indicate that all the three antibiotics tested are able to interfere with biofilm formation and EPS production by Salm. Typhimurium isolates. Significance and Impact of the Study:  The current study demonstrated that cefotaxime at sub‐MIC can be beneficial for the behaviour of pathogen Salm. Typhimurium in vitro.