Int J Mol Sci

Phage-displayed peptide selections generate complex repertoires of several hundred thousand peptides as revealed by next-generation sequencing (NGS). In repeated peptide selections, however, even in identical experimental in vitro conditions, only a very small number of common peptides are found. The repertoire complexities are evidence of the difficulty of distinguishing between effective selections of specific peptide binders to exposed targets and the potential high background noise. Such investigation is even more relevant when considering the plethora of in vivo expressed targets on cells, in organs or in the entire organism to define targeting peptide agents. In the present study, we compare the published NGS data of three peptide repertoires that were obtained by phage display under identical experimental in vitro conditions. By applying the recently developed tool PepSimili we evaluate the calculated similarities of the individual peptides from each of these three repertoires and perform their mappings on the human proteome. The peptide-to-peptide mappings reveal high similarities among the three repertoires, confirming the desired reproducibility of phage-displayed peptide selections

Differential aquaporin 4 expression during edema build-up and resolution phases of brain inflammation

<p>Abstract</p> <p>Background</p> <p>Vasogenic edema dynamically accumulates in many brain disorders associated with brain inflammation, with the critical step of edema exacerbation feared in patient care. Water entrance through blood-brain barrier (BBB) opening is thought to have a role in edema formation. Nevertheless, the mechanisms of edema resolution remain poorly understood. Because the water channel aquaporin 4 (AQP4) provides an important route for vasogenic edema resolution, we studied the time course of AQP4 expression to better understand its potential effect in countering the exacerbation of vasogenic edema.</p> <p>Methods</p> <p>Focal inflammation was induced in the rat brain by a lysolecithin injection and was evaluated at 1, 3, 7, 14 and 20 days using a combination of in vivo MRI with apparent diffusion coefficient (ADC) measurements used as a marker of water content, and molecular and histological approaches for the quantification of AQP4 expression. Markers of active inflammation (macrophages, BBB permeability, and interleukin-1β) and markers of scarring (gliosis) were also quantified.</p> <p>Results</p> <p>This animal model of brain inflammation demonstrated two phases of edema development: an initial edema build-up phase during active inflammation that peaked after 3 days (ADC increase) was followed by an edema resolution phase that lasted from 7 to 20 days post injection (ADC decrease) and was accompanied by glial scar formation. A moderate upregulation in AQP4 was observed during the build-up phase, but a much stronger transcriptional and translational level of AQP4 expression was observed during the secondary edema resolution phase.</p> <p>Conclusions</p> <p>We conclude that a time lag in AQP4 expression occurs such that the more significant upregulation was achieved only after a delay period. This change in AQP4 expression appears to act as an important determinant in the exacerbation of edema, considering that AQP4 expression is insufficient to counter the water influx during the build-up phase, while the second more pronounced but delayed upregulation is involved in the resolution phase. A better pathophysiological understanding of edema exacerbation, which is observed in many clinical situations, is crucial in pursuing new therapeutic strategies.</p

Comparative Evaluation of Reproducibility of Phage-Displayed Peptide Selections and NGS Data, through High-Fidelity Mapping of Massive Peptide Repertoires

Phage-displayed peptide selections generate complex repertoires of several hundred thousand peptides as revealed by next-generation sequencing (NGS). In repeated peptide selections, however, even in identical experimental in vitro conditions, only a very small number of common peptides are found. The repertoire complexities are evidence of the difficulty of distinguishing between effective selections of specific peptide binders to exposed targets and the potential high background noise. Such investigation is even more relevant when considering the plethora of in vivo expressed targets on cells, in organs or in the entire organism to define targeting peptide agents. In the present study, we compare the published NGS data of three peptide repertoires that were obtained by phage display under identical experimental in vitro conditions. By applying the recently developed tool PepSimili we evaluate the calculated similarities of the individual peptides from each of these three repertoires and perform their mappings on the human proteome. The peptide-to-peptide mappings reveal high similarities among the three repertoires, confirming the desired reproducibility of phage-displayed peptide selections

Comparative Evaluation of Reproducibility of Phage-Displayed Peptide Selections and NGS Data, through High-Fidelity Mapping of Massive Peptide Repertoires

Phage-displayed peptide selections generate complex repertoires of several hundred thousand peptides as revealed by next-generation sequencing (NGS). In repeated peptide selections, however, even in identical experimental in vitro conditions, only a very small number of common peptides are found. The repertoire complexities are evidence of the difficulty of distinguishing between effective selections of specific peptide binders to exposed targets and the potential high background noise. Such investigation is even more relevant when considering the plethora of in vivo expressed targets on cells, in organs or in the entire organism to define targeting peptide agents. In the present study, we compare the published NGS data of three peptide repertoires that were obtained by phage display under identical experimental in vitro conditions. By applying the recently developed tool PepSimili we evaluate the calculated similarities of the individual peptides from each of these three repertoires and perform their mappings on the human proteome. The peptide-to-peptide mappings reveal high similarities among the three repertoires, confirming the desired reproducibility of phage-displayed peptide selections

DNA Subtraction of Selected Phage Repertoires for Efficient Peptide Pathology Biomarker Identification in Neuroinflammation Multiple Sclerosis Model

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

Type 3 deiodinase expression in inflammatory spinal cord lesions in rat experimental autoimmune encephalomyelitis

BACKGROUND: We have shown substantial expression of type 3 deiodinase (D3, a major enzyme involved in the inactivation of thyroid hormone) in infiltrating leukocytes in several models of inflammation. Recently, thyroid hormone has been shown to improve remyelination in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. As induction of D3 may play an important role in decreasing local bioavailability of thyroid hormone at inflammation sites, we hypothesized that D3 is induced in spinal cord inflammatory lesions in EAE. METHODS: The aim of the study was to evaluate D3 expression in spinal cord inflammatory lesions of EAE Dark Agouti rats and to investigate D3 induction in activated monocytes. RESULTS: Here, we show marked expression of D3 by granulocytes and macrophages in spinal cord inflammatory lesions of EAE rats. We further confirm induction of D3 expression in vitro in monocytes that were activated toward proinflammatory or immunomodulatory phenotypes. CONCLUSIONS: We observed increased D3 expression both in spinal cord inflammatory lesions during EAE and in activated monocytes. Although increased D3 expression theoretically results in decreased triiodothyronine availability, it is unknown at present whether reduced local triiodothyronine concentrations are involved in impaired remyelination as observed during EA

Differential UDP-galactose-4′-epimerase (GALE) enzymatic activity and mRNA expression in the rat mammary gland during lactation

AbstractWe have investigated the UDP-galactose-4′-epimerase (GALE) enzymatic activity and mRNA expression in the rat mammary gland during lactation. We report a dramatic increase in the GALE enzymatic activity correlated with an increase in the mRNA transcript expression. These results indicate a transcriptional regulation of the enzyme during lactation in the rat mammary gland. Our data are of double interest for further investigation: first, the mammary gland provides a suitable model for the characterisation of the transcriptional regulation elements of GALE which are still unknown in mammals; second, GALE expression could help to compensate UDP-galactose deficiency in classic galactosaemia

Monitoring demyelination and remyelination by magnetization transfer imaging in the mouse brain at 9.4 T

none12siopenZaaraoui, W; Deloire, M; Merle, M; Girard, C; Raffard, G; Biran, M; Inglese, M; Petry, Kg; Gonen, O; Brochet, B; Franconi, Jm; Dousset, V.Zaaraoui, W; Deloire, M; Merle, M; Girard, C; Raffard, G; Biran, M; Inglese, MARIA MATILDE; Petry, Kg; Gonen, O; Brochet, B; Franconi, Jm; Dousset, V