RESUMO
Phage Display is a powerful method for the identification of peptide binding to targets of variable complexities and tissues, from unique molecules to the internal surfaces of vessels of living organisms. Particularly for in vivo screenings, the resulting repertoires can be very complex and difficult to study with traditional approaches. Next Generation Sequencing (NGS) opened the possibility to acquire high resolution overviews of such repertoires and thus facilitates the identification of binders of interest. Additionally, the ever-increasing amount of available genome/proteome information became satisfactory regarding the identification of putative mimicked proteins, due to the large scale on which partial sequence homology is assessed. However, the subsequent production of massive data stresses the need for high-performance computational approaches in order to perform standardized and insightful molecular network analysis. Systems-level analysis is essential for efficient resolution of the underlying molecular complexity and the extraction of actionable interpretation, in terms of systemic biological processes and pathways that are systematically perturbed. In this work we introduce PepSimili, an integrated workflow tool, which performs mapping of massive peptide repertoires on whole proteomes and delivers a streamlined, systems-level biological interpretation. The tool employs modules for modeling and filtering of background noise due to random mappings and amplifies the biologically meaningful signal through coupling with BioInfoMiner, a systems interpretation tool that employs graph-theoretic methods for prioritization of systemic processes and corresponding driver genes. The current implementation exploits the Galaxy environment and is available online. A case study using public data is presented, with and without a control selection.
RESUMO
To streamline in vivo biomarker discovery, we developed a suppression subtractive DNA hybridization technique adapted for phage-displayed combinatorial libraries of 12 amino acid peptides (PhiSSH). Physical DNA subtraction is performed in a one-tube-all-reactions format by sequential addition of reagents, producing the enrichment of specific clones of one repertoire. High-complexity phage repertoires produced by in vivo selections in the multiple sclerosis rat model (experimental autoimmune encephalomyelitis, EAE) and matched healthy control rats were used to evaluate the technique. The healthy repertoire served as a physical DNA subtractor from the EAE repertoire to produce the subtraction repertoire. Full next-generation sequencing (NGS) of the three repertoires was performed to evaluate the efficiency of the subtraction technique. More than 96% of the clones common to the EAE and healthy repertoires were absent from the subtraction repertoire, increasing the probability of randomly selecting various specific peptides for EAE pathology to about 70%. Histopathology experiments were performed to confirm the quality of the subtraction repertoire clones, producing distinct labeling of the blood-brain barrier (BBB) affected by inflammation among healthy nervous tissue or the preferential binding to IL1-challenged vs. resting human BBB model. Combining PhiSSH with NGS will be useful for controlled in vivo screening of small peptide combinatorial libraries to discover biomarkers of specific molecular alterations interspersed within healthy tissues.
RESUMO
A Reversed Phase-High Performance Liquid Chromatography/Diode Array Detection method was developed and validated for paracetamol quantification in cell culture fluid from an in vitro Blood Brain Barrier model. The chromatographic method and sample preparation were developed using only aqueous solvents. The column was a XTerra RP18 150 × 4.6mm, 3.5 µm with a guard column XTerra RP18 20 × 4.6 mm, 3.5 µm at 35 °C and the mobile phase was composed by 100% formate buffer 20 mM at pH 4 and flow rate was set at 1 mL/min. The detection was at 242 nm. The sample was injected at 10 µL. Validation was performed using the accuracy profile approach. The analytical procedure was validated with the acceptance limits at ± 10% over a range of concentration from 1 to 58 mg L(-1). The procedure was then used in routine to determine paracetamol concentration in a brain blood barrier in vitro model. Application of the Unither paracetamol formulation in Blood Brain Barrier model allowed the determination and comparison of the transcellular passage of paracetamol at 37 °C and 4 °C, that excludes paracellular or non specific leakage.
