Detection of virulence and pathogenicity genes in selected phytopathovars

Plant pathogenic organisms are known to infect host cell using various range of secretory proteins. Amongst all other secretion systems, type III secretion system (T3SS) is a key mechanism for bacterial pathogenesis for establishing and maintaining infection into the host. Expression levels of seven genes viz. avrXacE1, avrXacE2, hpaA and hrpG along with bacterial endogenous control lrp (leucine-responsive protein) were studied. The pathogenic organisms selected for the present study includes Enterobacter cloacae, Enterobacter spp., Pantoea ananatis, Xanthomonas campestris pv. Citri, Pantoea agglomerans, Ochrobactrum anthropi and Erwinia chrysanthemi. P. agglomerans and Enterobacter spp. gave high expression of above-mentioned virulence genes compared to Xanthomonas, while E. cloacae and P. ananatis showed similar expression with that of Xanthomonas. The detailed relationship of the expression profiles with respect to the selected organisms is discussed.     

Quantitation of wall teichoic acid in Staphylococcus aureus by direct measurement of monomeric units using LC-MS/MS

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) has created an urgent need for new therapeutic agents capable of combating this threat. We have previously reported on the discovery of novel inhibitors targeting enzymes involved in the biosynthesis of wall teichoic acid (WTA) and demonstrated that these agents can restore β-lactam efficacy against MRSA. In those previous reports pathway engagement of inhibitors was demonstrated by reduction in WTA levels measured by polyacrylamide gel electrophoresis. To enable a more rigorous analysis of these inhibitors we sought to develop a quantitative method for measuring whole-cell reductions in WTA. Herein we describe a robust methodology for hydrolyzing polymeric WTA to the monomeric component ribitol-N-acetylglucosamine coupled with measurement by LC-MS/MS. Critical elements of the protocol were found to include the time and temperature of hydrofluoric acid-mediated hydrolysis of polymeric WTA and optimization of these parameters is fully described. Most significantly, the assay enabled accurate and reproducible measurement of depletion EC_50s for tunicamycin and representatives from the novel class of TarO inhibitors, the tarocins. The method described can readily be adapted to quantifying levels of WTA in tissue homogenates from a murine model of infection, highlighting the applicability for both in vitro and in vivo characterizations.     

Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation

There is a groundswell of interest in using genetically engineered sensor bacteria to study gut microbiota pathways, and diagnose or treat associated diseases. Here, we computationally identify the first biological thiosulfate sensor and an improved tetrathionate sensor, both two‐component systems from marine Shewanella species, and validate them in laboratory Escherichia coli. Then, we port these sensors into a gut‐adapted probiotic E. coli strain, and develop a method based upon oral gavage and flow cytometry of colon and fecal samples to demonstrate that colon inflammation (colitis) activates the thiosulfate sensor in mice harboring native gut microbiota. Our thiosulfate sensor may have applications in bacterial diagnostics or therapeutics. Finally, our approach can be replicated for a wide range of bacterial sensors and should thus enable a new class of minimally invasive studies of gut microbiota pathways.     

Fully activated MEK1 exhibits compromised affinity for binding of allosteric inhibitors U0126 and PD0325901

Kinases catalyze the transfer of γ-phosphate from ATP to substrate protein residues triggering signaling pathways responsible for a plethora of cellular events. Isolation and production of homogeneous preparations of kinases in their fully active forms is important for accurate in vitro measurements of activity, stability, and ligand binding properties of these proteins. Previous studies have shown that MEK1 can be produced in its active phosphorylated form by coexpression with RAF1 in insect cells. In this study, using activated MEK1 produced by in vitro activation by RAF1 (pMEK1(in?vitro)), we demonstrate that the simultaneous expression of RAF1 for production of activated MEK1 does not result in stoichiometric phosphorylation of MEK1. The pMEK1(in vitro) showed higher specific activity toward ERK2 protein substrate compared to the pMEK1 that was activated via coexpression with RAF1 (pMEK1(in situ)). The two pMEK1 preparations showed quantitative differences in the phosphorylation of T-loop residue serine 222 by Western blotting and mass spectrometry. Finally, pMEK1(in vitro) showed marked differences in the ligand binding properties compared to pMEK1(in?situ). Contrary to previous findings, pMEK1(in vitro) bound allosteric inhibitors U0126 and PD0325901 with a significantly lower affinity than pMEK1(in situ) as well as its unphosphorylated counterpart (npMEK1) as demonstrated by thermal-shift, AS-MS, and calorimetric studies. The differences in inhibitor binding affinity provide direct evidence that unphosphorylated and RAF1-phosphorylated MEK1 form distinct inhibitor sites.     

Transcervical Ultrasonography Is Feasible to Visualize and Evaluate Base of Tongue Cancers

Background

Base of tongue (BOT) is a difficult subsite to examine clinically and radiographically. Yet, anatomic delineation of the primary tumor site, its extension to adjacent sites or across midline, and endophytic vs. exophytic extent are important characteristics for staging and treatment planning. We hypothesized that ultrasound could be used to visualize and describe BOT tumors.

Methods

Transcervical ultrasound was performed using a standardized protocol in cases and controls. Cases had suspected or confirmed BOT malignancy. Controls were healthy individuals without known malignancy.

Results

100% of BOT tumors were visualized. On ultrasound BOT tumors were hypoechoic (90.9%) with irregular margins (95.5%). Ultrasound could be used to characterize adjacent site involvement, midline extent, and endophytic extent, and visualize the lingual artery. No tumors were suspected for controls.

Conclusions

Ultrasonography can be used to transcervically visualize BOT tumors and provides clinically relevant characteristics that may not otherwise be appreciable.