Development of indole chemistry to label tryptophan residues in protein for determination of tryptophan surface accessibility

Solvent accessibility can be used to evaluate protein structural models, identify binding sites, and characterize protein conformational changes. The differential modification of amino acids at specific sites enables the accessible surface residues to be identified by mass spectrometry. Tryptophan residues within proteins can be differentially labeled with halocompounds by a photochemical reaction. In this study, tryptophan residues of carbonic anhydrase are reacted with chloroform, 2,2,2-trichloroethanol (TCE), 2,2,2-trichloroacetate (TCA), or 3-bromo-1-propanol (BP) under UV irradiation at 280 nm. The light-driven reactions with chloroform, TCE, TCA, and BP attach a formyl, hydroxyethanone, carboxylic acid, and propanol group, respectively, onto the indole ring of tryptophan. Trypsin and chymotrypsin digests of the modified carbonic anhydrase are used to map accessible tryptophan residues using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Tryptophan reactivity is determined by identifying peptides with tryptophan residues modified with the appropriate label. The reactivity is calculated from the frequency that the modification is identified and a semiquantitative measure of the amount of products formed. Both of these measures of tryptophan reactivity correlate significantly with the accessible surface area of tryptophan residues in carbonic anhydrase determined from the X-ray crystal structure. Therefore the photochemical reaction of halocompounds with tryptophan residues in carbonic anhydrase indicates the degree of solvent accessibility of these residues.     

A Pseudomonas aeruginosa TIR effector mediates immune evasion by targeting UBAP1 and TLR adaptors

Bacterial pathogens often subvert the innate immune system to establish a successful infection. The direct inhibition of downstream components of innate immune pathways is particularly well documented but how bacteria interfere with receptor proximal events is far less well understood. Here, we describe a Toll/interleukin 1 receptor (TIR) domain‐containing protein (PumA) of the multi‐drug resistant Pseudomonas aeruginosa PA7 strain. We found that PumA is essential for virulence and inhibits NF‐κB, a property transferable to non‐PumA strain PA14, suggesting no additional factors are needed for PumA function. The TIR domain is able to interact with the Toll‐like receptor (TLR) adaptors TIRAP and MyD88, as well as the ubiquitin‐associated protein 1 (UBAP1), a component of the endosomal‐sorting complex required for transport I (ESCRT‐I). These interactions are not spatially exclusive as we show UBAP1 can associate with MyD88, enhancing its plasma membrane localization. Combined targeting of UBAP1 and TLR adaptors by PumA impedes both cytokine and TLR receptor signalling, highlighting a novel strategy for innate immune evasion.     

Fertilization effects with P on accumulated leaf area duration,biomass and yield of three cultivars of maize in Toluca,Mexico

The effect of six phosphorus levels (0, 40, 80, 120, 160 and 200 kg/ha) on the duration of cumulative leaf area, biomass and agronomic yield was determined in the maize cultivars: Amarillo Almoloya, Cacahuacintle and Condor in 2010 and 2011. Such cultivars were sown in the Cerrillo Piedras Blancas Mexico. A completely randomized complete block design with factorial arrangement was utilized. High phosphorus levels (120, 160 and 200 kg/ha) positively affected the duration of cumulative leaf area; greatest values were obtained in Cacahuacintle. A greater duration of accumulated leaf area contributes to determine high values of biomass accumulation and grain yield in this cultivar. Leaf area duration appeared to be a useful tool for evaluating different genotypes in a given environment.     

Quantitative biophysical metrics for rapid evaluation of ovarian cancer metastatic potential

Ovarian cancer is routinely diagnosed long after the disease has metastasized through the fibrous submesothelium. Despite extensive research in the field linking ovarian cancer progression to increasingly poor prognosis, there are currently no validated cellular markers or hallmarks of ovarian cancer that can predict metastatic potential. To discern disease progression across a syngeneic mouse ovarian cancer progression model, here we fabricated extracellular matrix mimicking suspended fiber networks: cross-hatches of mismatch diameters for studying protrusion dynamics, aligned same diameter networks of varying interfiber spacing for studying migration, and aligned nanonets for measuring cell forces. We found that migration correlated with disease while a force-disease biphasic relationship exhibited F-actin stress fiber network dependence. However, unique to suspended fibers, coiling occurring at the tips of protrusions and not the length or breadth of protrusions displayed the strongest correlation with metastatic potential. To confirm that our findings were more broadly applicable beyond the mouse model, we repeated our studies in human ovarian cancer cell lines and found that the biophysical trends were consistent with our mouse model results. Altogether, we report complementary high throughput and high content biophysical metrics capable of identifying ovarian cancer metastatic potential on a timescale of hours.     

A correction to the sequence of the alpha chains of horse haemoglobin

An atomic model of horse aquomethaemoglobin extensively refined against 2.0 Å X-ray data suggested that an Ala and a Gly residue in the published sequence of the α chain were interchanged. Chemical analysis has confirmed our interpretation of the model.