Previous studies have shown that a mitogen activated protein (MAP) kinase (MEK)-independent signaling pathway is required by activated Raf or fibroblast-derived growth factor (FGF) for the differentiation of rat hippocampal neuronal H19-7 cells. We now demonstrate that both Raf and FGF similarly induce prolonged transcription and translation of the immediate early gene pip92 in the absence of activation of the MAP kinases (MAPKs) ERK1 and ERK2. To determine the mechanism by which this occurs and to identify novel Rafactivated signaling pathways, we investigated the induction of the pip92 promoter by both FGF and an estradiol-activated Raf-1-estrogen receptor fusion protein (⌬Raf-1:ER) in H19-7 cells. Deletion analysis of the pip92 promoter indicated that activation by the MAPK-independent pathway occurs primarily within the region containing a serum response element (SRE). Further analysis of the SRE by using a heterologous thymidine kinase promoter showed that both an Ets and CArG-like site are required. Elk1, which binds to the Ets site, was phosphorylated both in vitro and in vivo by the MAPK-independent pathway, and phosphorylation of an Elk1-GAL4 fusion protein by this pathway was sufficient for transactivation. Finally, at least two Elk1 kinases were fractionated by gel filtration, and analysis by an in-gel kinase assay revealed at least three novel Raf-activated Elk1 kinases. These results indicate that both FGF and Raf activate MAPK-independent kinases that can stimulate Elk1 phosphorylation and immediate early gene transcription.

Research in microfluidic biosensors has led to dramatic improvements in sensitivities. Very few examples of these devices have been commercially successful, keeping this methodology out of the hands of potential users. In this study, we developed a method to fabricate a flexible microfluidic device containing electrowetting valves and electrochemical transduction. The device was designed to be amenable to a roll-to-roll manufacturing system, allowing a low manufacturing cost. Microchannels with high fidelity were structured on a PET film using UV-NanoImprint Lithography (UV-NIL). The electrodes were inkjet-printed and photonically sintered on second flexible PET film. The film containing electrodes was bonded directly to the channel-containing layer to form sealed fluidic device. Actuation of the multivalve system with food dye in PBS buffer was performed to demonstrate automated fluid delivery. The device was then used to detect Salmonella in a liquid sample.

The application of bacteriophage combined with the use of magnetic separation techniques has emerged as a valuable tool for the sensitive identification and detection of bacteria. In this study, bacteriophage T7 labelled magnetic beads were developed for the detection of viable bacterial cells. Fusion of the biotin acceptor peptide (BAP) with the phage capsid protein gene and the insertion of the biotin ligase (BirA) gene enabled the display of the BAP ligand and the expression protein BirA during the replication cycle of phage infection. The replicated Escherichia coli specific bacteriophage was biotinylated in vivo and coated on magnetic beads via streptavidin-biotin interaction. Immobilization efficiency of the recombinant phage was investigated on magnetic beads and the phage-bead complex was evaluated by detecting E. coli from inoculated broth. When compared to the wild type phage, the recombinant phage T7birA-bap had a high immobilization density on streptavidin-coated magnetic beads and could capture 86.2% of E. coli cells from broth within 20 min. As this phage-based biomagnetic detection approach provided a low detection limit of 10(2) CFU mL(-1) without pre-enrichment, we believe this assay could be further developed to detect other bacteria of interest by applying host-specific phages. This would be of particular use in detecting bacteria which are difficult to grow or replicate slowly in culture.

In this study, an enzyme-based electrochemical method was developed for the detection of Escherichia coli (E. coli) using the T7 bacteriophages engineered with lacZ operon encoding for beta-galactosidase (β-gal). The T7 phages can infect E. coli, and have the ability to trigger the overexpression of β-gal during the infection of E. coli. The use of the engineered phages resulted in a more sensitive detection of E. coli by (1) overexpression of β-gal in E. coli during the specific infection and (2) release of the endogenous intracellular β-gal from E. coli following infection. The endogenous and phage-induced β-gal was detected using the electrochemical method with 4-aminophenyl-β-galactopyranoside (PAPG) as a substrate. The β-gal catalyzed PAPG to an electroactive species p-aminophenol (PAP) which could be monitored on an electrode. The electrochemical signal was proportional to the concentration of E. coli in the original sample. We demonstrated the application of our strategy in aqueous samples (drinking water, apple juice, and skim milk). Using this method, we were able to detect E. coli at the concentration of approximately 10 CFU/mL in these aqueous samples in 3 h and 10 CFU/mL after 7 h. This strategy has the potential to be extended to detect different bacteria using specific bacteriophages engineered with gene encoding for appropriate enzymes.

BACKGROUND Although decoding the molecular mechanisms underlying insecticide resistance has often proven difficult, recent progress has revealed that specific mutations in the ryanodine receptor (RyR) of the diamondback moth, Plutella xylostella, can confer resistance to diamide insecticides. The extent to which specific RyR mutations contribute to the diamide resistance phenotype, the associated genetic traits and fitness costs remain limited. RESULTS Three field‐evolved PxRyR mutations (G4946E, I4790 M, and I4790 K) were respectively introgressed into a common susceptible background strain (IPP‐S) of P. xylostella with marker‐assisted backcrossing. The mutations alone can result in moderate to high levels of resistance to five commercial diamides (flubendiamide, chlorantraniliprole, cyantraniliprole, tetraniliprole, and cyclaniliprole), and the resistance intensity mediated by the three mutations was hierarchical in order of I4790 K (1199‐ to >2778‐fold) > G4946E (39‐ to 739‐fold) > I4790 M (16‐ to 57‐fold). Flubendiamide resistance was autosomal and incompletely recessive, and was significantly linked with the introgressed mutations in the three constructed strains. In addition, the resistance levels to flubendiamide of hybrid progeny from any two resistant strains fell in between the status of their parents. Furthermore, by comparing the net replacement rate, the fitness of 4946E, 4790 M and 4790 K strains were 0.77, 0.93 and 0.92 relative to the IPP‐S strain, respectively. CONCLUSION Three independent PxRyR mutations confer varying degrees of resistance to diamides in P. xylostella. Among the three mutations, I4790 K confers highest levels of resistance (> 1000‐fold) to all five commercial diamides. The findings can guide resistance management practices for diamides in P. xylostella and other arthropods.

The monitoring of drinking water for indicators of fecal contamination is crucial for ensuring a safe supply.

An electrochemical method based on redox cycling combined with immunomagnetic separation and pre-concentration was developed for rapid and sensitive detection of Salmonella. Electrochemical methods for the detection of bacteria offer the advantages of instant quantification with minimal equipment. Unfortunately, the limits of detection are often poor compare to other transduction methods such as fluorescence and chemiliuminescence. We demonstrated an electrochemical method which is both rapid and has a low limit of detection. A two-step strategy, which included immunomagentic pre-concentration and redox cycling was used to amplify the signal. Magnetic beads modified with anti-Salmonella antibodies were used for separation and pre-concentration of Salmonella from phosphate buffered saline (PBS) and agricultural water. Then anti-Salmonella antibodies conjugated with alkaline phosphatase were employed for labeling the Salmonella which had been captured by magnetic beads. Alkaline phosphatase (ALP) catalyzed the substrate Lascorbic acid 2-phosphate (AAP) to electroactive species L-ascorbic acid (AA) while tris(2-carboxyethyl)phosphine (TCEP) facilitated the regeneration of AA on the gold electrode to form redox cycling resulting in an amplified signal. Under the optimal conditions, the Salmonella in PBS buffer as well as in agricultural water were detected. The limit of detection of this approach was approximately 7.6 × 10 2 CFU/mL and 6.0 × 10 2 CFU/mL in PBS buffer and agricultural water, respectively, without pre-enrichment in 3 hours. When the agricultural water has been pre-enriched for 4 hours, the limit of detection was approximately 10 CFU/mL.

Ease of use, low cost, and convenient transport are the key requirements for a commercial bacteria detection kit designed for resource-limited settings. Here, we report the colorimetric detection of Escherichia coli (E. coli) in food samples using freeze-dried engineered bacteriophages (phages). In this approach, we have engineered T7 phages to carry the lacZ operon driven by T7 promoter to overexpress reporter enzymes. The engineered phages were freeze-dried in a water-soluble polymer for storage and transportation. When used for the detection of E. coli cells, the intracellular enzyme [β-galactosidase (β-gal)] was overexpressed and released into the surrounding media, providing an enzyme-amplified colorimetric signal. Using this strategy, we were able to detect E. coli cells at the concentration of 10 CFU mL in food samples without the need for sophisticated instruments or skilled operators.