A highly multiplexed quantitative phosphosite assay for biology and preclinical studies

Reliable methods to quantify dynamic signaling changes across diverse pathways are needed to better understand the effects of disease and drug treatment in cells and tissues but are presently lacking. Here, we present SigPath, a targeted mass spectrometry (MS) assay that measures 284 phosphosites in 200 phosphoproteins of biological interest. SigPath probes a broad swath of signaling biology with high throughput and quantitative precision. We applied the assay to investigate changes in phospho‐signaling in drug‐treated cancer cell lines, breast cancer preclinical models, and human medulloblastoma tumors. In addition to validating previous findings, SigPath detected and quantified a large number of differentially regulated phosphosites newly associated with disease models and human tumors at baseline or with drug perturbation. Our results highlight the potential of SigPath to monitor phosphoproteomic signaling events and to nominate mechanistic hypotheses regarding oncogenesis, response, and resistance to therapy.     

Influences of dietary soy isoflavones on metabolism but not nociception and stress hormone responses in ovariectomized female rats

Background  

Isoflavones, the most abundant phytoestrogens in soy foods, are structurally similar to 17beta-estradiol. Few studies have examined the nociception and stress hormone responses after consumption of soy isoflavones.     

Total antioxidant capacity – a novel early bio-chemical marker of oxidative stress in HIV infected individuals

Background  

Oxidative stress induced by the production of reactive oxygen species may play a critical role in the stimulation of HIV replication and the development of immunodeficiency. This study was conducted as there are limited and inconclusive studies on the significance of a novel early marker of oxidative stress which can reflect the total antioxidant capacity in HIV patients,     

Evolution of cytochrome P-450 proteins [published erratum appears in Mol Biol Evol 1988 Mar;5(2):199]

Thirty-four cytochrome P-450 sequences from one bacterial and six vertebrate species have been aligned with the aid of a computer alignment algorithm. Phylogenetic trees were constructed using the unweighted-pair-group and neighbor-joining methods. The two trees differed at only a single branch point near the base of the tree. The cytochrome P-450 superfamily of proteins clustered into eight families and contained 16 gene-duplication events. The first gene duplication occurred approximately 1,360 Myr before the present (Mybp) and gave rise to cytochrome P-450s found in two different cellular organelles, the mitochondria and the endoplasmic reticulum. Both groups utilize cholesterol or its metabolites as substrates, implying that cholesterol existed greater than 1,360 Mybp. The fourth gene duplication (approximately 900 Mybp) gave rise to the drug-metabolizing P-450s. These proteins aid in the detoxification of foreign chemicals, as opposed to the metabolism of endogenous compounds. The importance of the capacity to metabolize drugs is reflected in 11 further gene duplications occurring in this lineage. The first occurred approximately 800 Mybp and gave rise to the two major P-450 families, the phenobarbital and 3-methylcholanthrene families. An apparent increase in the rate of cytochrome P-450 evolution is noted between the bird-mammal divergence (300 Mybp) and the mammalian radiation (75 Mybp).      

Development of an optimal sampling protocol for millipedes (Diplopoda)

This study determined a millipede sampling protocol that was efficient, replicable, and thorough, by testing commonly used methods (pitfall trapping, hand collection, and leaf litter collection with Berlese extraction) at sites where the fauna had been intensively sampled, so that the results of different sampling protocols (‘experimental sampling’) could be compared to the previously known fauna of each site (determined by ‘baseline sampling’). Two methods of collecting leaf litter were compared. One method allowed the collector to choose the locations from which to sample leaf litter, while the second method used randomly generated locations. Experimental sampling took place at both an upland site (Swallow Cliff Woods, Cook Co, IL) and a wetland site (Cedarburg Bog, Ozaukee Co, WI) where reliable baseline data was available. The optimal protocol for millipedes was found to consist of a combination of the hand collection and the a priori litter methods. In studies where there are constraints on time or resources devoted to sampling or processing, this protocol can be reduced to the single most effective method, hand collection. Overall, the a priori litter collection method was more effective than the random litter collection method.