SORTCERY—A High–Throughput Method to Affinity Rank Peptide Ligands

Uncovering the relationships between peptide and protein sequences and binding properties is critical for successfully predicting, re-designing and inhibiting protein–protein interactions. Systematically collected data that link protein sequence to binding are valuable for elucidating determinants of protein interaction but are rare in the literature because such data are experimentally difficult to generate. Here we describe SORTCERY, a high-throughput method that we have used to rank hundreds of yeast-displayed peptides according to their affinities for a target interaction partner. The procedure involves fluorescence-activated cell sorting of a library, deep sequencing of sorted pools and downstream computational analysis. We have developed theoretical models and statistical tools that assist in planning these stages. We demonstrate SORTCERY's utility by ranking 1026 BH3 (Bcl-2 homology 3) peptides with respect to their affinities for the anti-apoptotic protein Bcl-x[subscript L]. Our results are in striking agreement with measured affinities for 19 individual peptides with dissociation constants ranging from 0.1 to 60 nM. High-resolution ranking can be used to improve our understanding of sequence–function relationships and to support the development of computational models for predicting and designing novel interactions.National Institutes of Health (U.S.) (Award GM096466)German Academic Scholarship Foundation (Grant RE 3111/1-1

Generating High-Accuracy Peptide-Binding Data in High Throughput with Yeast Surface Display and SORTCERY

Library methods are widely used to study protein-protein interactions, and high-throughput screening or selection followed by sequencing can identify a large number of peptide ligands for a protein target. In this chapter, we describe a procedure called "SORTCERY" that can rank the affinities of library members for a target with high accuracy. SORTCERY follows a three-step protocol. First, fluorescence-activated cell sorting (FACS) is used to sort a library of yeast-displayed peptide ligands according to their affinities for a target. Second, all sorted pools are deep sequenced. Third, the resulting data are analyzed to create a ranking. We demonstrate an application of SORTCERY to the problem of ranking peptide ligands for the anti-apoptotic regulator Bcl-xL.National Institute of General Medical Sciences (U.S.) (Award GM096466

Identification of Habitat-Specific Biomes of Aquatic Fungal Communities Using a Comprehensive Nearly Full-Length 18S rRNA Dataset Enriched with Contextual Data

Molecular diversity surveys have demonstrated that aquatic fungi are highly diverse, and that they play fundamental ecological roles in aquatic systems. Unfortunately, comparative studies of aquatic fungal communities are few and far between, due to the scarcity of adequate datasets. We combined all publicly available fungal 18S ribosomal RNA (rRNA) gene sequences with new sequence data from a marine fungi culture collection. We further enriched this dataset by adding validated contextual data. Specifically, we included data on the habitat type of the samples assigning fungal taxa to ten different habitat categories. This dataset has been created with the intention to serve as a valuable reference dataset for aquatic fungi including a phylogenetic reference tree. The combined data enabled us to infer fungal community patterns in aquatic systems. Pairwise habitat comparisons showed significant phylogenetic differences, indicating that habitat strongly affects fungal community structure. Fungal taxonomic composition differed considerably even on phylum and class level. Freshwater fungal assemblage was most different from all other habitat types and was dominated by basal fungal lineages. For most communities, phylogenetic signals indicated clustering of sequences suggesting that environmental factors were the main drivers of fungal community structure, rather than species competition. Thus, the diversification process of aquatic fungi must be highly clade specific in some cases.The combined data enabled us to infer fungal community patterns in aquatic systems. Pairwise habitat comparisons showed significant phylogenetic differences, indicating that habitat strongly affects fungal community structure. Fungal taxonomic composition differed considerably even on phylum and class level. Freshwater fungal assemblage was most different from all other habitat types and was dominated by basal fungal lineages. For most communities, phylogenetic signals indicated clustering of sequences suggesting that environmental factors were the main drivers of fungal community structure, rather than species competition. Thus, the diversification process of aquatic fungi must be highly clade specific in some cases

Application of hot water and cold air to reduce bacterial contamination on broiler carcasses

Two physical treatments (heat via water bath and cold air) with various temperatures (20/70/75/80°C and − 80/−90°C) and exposure times (20, 30, 40 s) were carried out to identify a decontaminating effect on zoonotic pathogens on broiler carcasses. Subsequently, carcasses were analyzed for thermotolerant Campylobacter (C.), Salmonella, Escherichia (E.). coli and total colony count (TCC). Moreover, for the hot water treatment, qPCR with viable/dead differentiation (v-qPCR) was applied to detect viable but non-culturable cells (VBNC) of Campylobacter, referred to as intact but putatively infectious units (IPIU). Hot water immersion was tested on carcasses inoculated with C. jejuni and Salmonella, while cold air treatment was evaluated for naturally contaminated carcasses of broiler flocks colonized with Campylobacter. For hot water treatment, the statistically significant reducing effect was about 1 log10 CFU/ml for both Salmonella and Campylobacter for 70–80°C and 20/30 s treatments. The effect of heat treatment for Campylobacter was smaller when samples were analyzed with v-qPCR with reductions of 0.5–0.8 log10 IPIU/ml in mean. Cold air treatments at −90°C were effective in reducing the mean contamination level of Campylobacter by 0.4–0.5 log10 CFU/ml at all exposure times (p < 0.05). Hot water treatments showed a decreasing trend on TCC by 0.6–0.9 log10 CFU/ml (p < 0.05). TCC counts were not significantly affected by cold air treatment. For E. coli no statistically significant reductions were observed by hot water treatment. The cold air treatment at −90°C for 20 and 40 s led to a reduction of E. coli by 0.4 and 0.8 log10 CFU/ml (p < 0.05), respectively. Treatment of carcasses with higher bacterial levels tended to show higher reduction. The research demonstrated that the efficacy of physical treatments for decontamination of broiler carcasses was more pronounced for hot water immersion than for cold air exposure. In conclusion, the results shed light on the potential application of these physical treatments in practice to reduce the quantitative load of contaminating pathogens to enhance food safety in the broiler meat production

Prominent oncogenic roles of EVI1 in breast carcinoma

Overexpression of the EVI1 oncogene is associated typically with aggressive myeloid leukemia, but is also detectable in breast carcinoma (BC) where its contributions are unexplored. Analyzing a tissue microarray of 608 BC patient specimens, we documented EVI1 overexpression in both estrogen receptor-positive (ER+) and estrogen receptor-negative (ER-) BC. Here we report prognostic relevance of EVI1 overexpression in triple-negative BC (TNBC) but not in the HER2-positive BC subset. In human breast cancer cells, EVI1 silencing reduced proliferation, apoptosis resistance and tumorigenicity, effects rescued by estrogen supplementation in ER+ BC cells. Estrogen addition restored ERK phosphorylation in EVI1-silenced cells, suggesting that EVI1 and estradiol signaling merge in MAPK activation. Conversely, EVI1 silencing had no effect on consitutive ERK activity in HER2+ BC cells. Microarray analyses revealed G-protein coupled receptor (GPR) signaling as a prominent EVI1 effector mechanism in BC. Among others, the GPR54-ligand KISS1 was identified as a direct transcriptional target of EVI1, which together with other EVI1-dependent cell motility factors such as RHOJ regulated BC cell migration. Overall, our results establish the oncogenic contributions of EVI1 in ER- and HER2-negative subsets of breast cancer