The peptide secreted at the water to land transition in a model amphibian has antioxidant effects

In addition to the morphophysiological changes experienced by amphibians during metamorphosis, they must also deal with a different set of environmental constraints when they shift from the water to the land. We found that Pithecopus azureus secretes a single peptide ([M + H]+ = 658.38 Da) at the developmental stage that precedes the onset of terrestrial behaviour. De novo peptide and cDNA sequencing revealed that the peptide, named PaT-2, is expressed in tandem and is a member of the tryptophyllins family. In silico studies allowed us to identify the position of reactive sites and infer possible antioxidant mechanisms of the compounds. Cell-based assays confirmed the predicted antioxidant activity in mammalian microglia and neuroblast cells. The potential neuroprotective effect of PaT-2 was further corroborated in FRET-based live cell imaging assays, where the peptide prevented lipopolysaccharide-induced ROS production and glutamate release in human microglia. In summary, PaT-2 is the first peptide expressed during the ontogeny of P. azureus , right before the metamorphosing froglet leaves the aquatic environment to occupy terrestrial habitats. The antioxidant activity of PaT-2, predicted by in silico analyses and confirmed by cell-based assays, might be relevant for the protection of the skin of P. azureus adults against increased O 2 levels and UV exposure on land compared with aquatic environments.

Peptide Conjugate on Multilayer Graphene Oxide Film for the Osteogenic Differentiation of Human Wharton’s Jelly-Derived Mesenchymal Stem Cells

Graphene oxide (GO) is extensively studied as a template material for mesenchymal stem cell application due to its two-dimensional nature and unique functionalization chemistries. Herein, a new type of peptide-conjugated multilayer graphene oxide (peptide/m-GO film) was fabricated and used as biomaterial for culturing human Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs). The characterization of the peptide/m-GO films was performed, and the biocompatibility of the WJ-MSCs on the peptide/m-GO films was investigated. The results demonstrated that the peptide conjugate on the m-GO film did not hamper the normal growth of WJ-MSCs but supported the growth of WJ-MSCs after the 6-day culture period. In addition, the osteogenic differentiation of WJ-MSCs on the peptide/m-GO films was enhanced as compared with the parent m-GO film. Therefore, such peptide-conjugated m-GO films could provide a highly biocompatible and multifunctional 2D material to tailor the potential application of WJ-MSCs in bone tissue regeneration.

Domain I of β2GPI is capable of blocking serum IgA antiphospholipid antibodies binding in vitro: an effect enhanced by PEGylation

Objectives This study aims to inhibit antiphospholipid syndrome (APS) serum derived IgA anti-beta-2-glycoprotein I (aβ2GPI) binding using Domain I (DI). Methods Serum from 13 APS patients was tested for IgA aβ2GPI and Anti-Domain I. Whole IgA was purified by peptide M affinity chromatography from positive serum samples. Serum was tested for IgA aβ2GPI binding in the presence and absence of either DI or of two biochemically modified variants containing either 20 kDa of poly(ethylene glycol) (PEG) or 40 kDa of PEG. Results Significant inhibition with DI was possible with average inhibition of 23% ( N = 13). Further inhibitions using 20 kDa PEG-DI and 40 kDa PEG-DI variants showed significant inhibition ( p = 0.0001) with both the 40 kDa PEG-DI and 20 kDa PEG-DI variants showing increased inhibition compared with DI alone ( p = 0.0001 and p = 0.001, n = 10). Conclusions Inhibition of IgA aβ2GPI by DI is possible and can be enhanced by biochemical modification in a subset of patients.

Quantitative Proteomics Identify Urinary Hgfl Protein As a Potential Marker for the Development of Chronic Kidney Disease in Sickle Cell Disease Patients

BACKGROUND: Chronic kidney disease (CKD) is common in patients with sickle cell disease (SCD). However, the progression of CKD in SCD and factors associated with such progression remain poorly defined. Liquid chromatography mass spectrometry (LC-MS) based quantitative proteomics has become a highly potent method for biomarker discovery due to growing capabilities for broad proteome coverage and good accuracy and precision in quantification. OBJECTIVES: The purpose of this study was to identify the potential markers associated with CKD progression in patients with SCD using quantitative proteomics. METHODS: Urine samples were collected from healthy controls and SCD patients with different CKD stages from University of Illinois at Chicago (UIC). Mass-spectrometry analysis was performed on an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific) coupled to a Prominence Nano LC (Shimadzu) using the Xcalibur version 2.7.0 (Thermo Scientific). Proteome Discoverer 1.4 and SIEVE 2.1 programs were used for protein identification and label-free quantification. Heavy isotope labeled peptide EDQTSPAPGLR(13C6, 15N) was used as an internal standard for high resolution/selected ion monitoring (HR/SIM) analysis of HGFL. Urinary HGFL protein together wiht creatinine and albumin were also measured by ELISA. RESULTS: Since glomerular hyperfiltration is an early stage of renal dysfunction. We performed label-free quantitative proteomic analysis for urine samples collected from SCD patients with hyperfiltration (N=3) and normal (N=3). Hepatocyte growth factor-like (HGFL) protein was found to be significantly downregulated (5.52-fold, p=8.05 × 10-5) in samples with glomerular hyperfiltration compared to normal group. Next, we developed a high resolution/selected ion monitoring (HR/SIM) method by measuring the HGFL peptide (m/z 585.79) with isotope labeled-HGFL peptide (m/z 590.80) as internal standard (IS). HR/SIM quantification was performed for 19 urine samples from SCD patients and 12 urine samples from healthy controls. HGFL levels were found to be significantly downregulated (p=0.0084) in the SCD urine samples compared to samples from healthy controls (Figure 1). To further assess the correlation between HGFL level and CDK stage, we expanded the analysis to SCD patients with different CKD stage ranging from 0 to 5 and 19 healthy individuals by ELISA. The result confirmed the finding of HR/SIM quantification, moreover, showed that urinary HGFL level highly correlated with CKD stage (r= ̶ 0.4106, p=0.002, Figure 1) and showed high sensitivity and specificity by Receiver Operating Characteristic (ROC) curve analysis (AUC=0.78). CONCLUSIONS: HGFL protein has been identified as a negative regulator of phosphatidylinositol 3-kinase (PI3K), and PI3K/Akt pathway was found to be activated in the progress of CKD. Therefore, the decrease of HGFL level in urines from SCD patients may indicate the development of CKD. Combination of LC-MS based quantitative proteomics and ELISA validation is an useful approach for biomarker discovery. ACKNOWLEDGMENTS: This work was supported by NIH Research Grants 1P50HL118006, 1R01HL125005, 5G12MD007597 and K23HL125984. The content is solely the responsibility of the authors and does not necessarily represent the official view of NHLBI, NIMHD or NIH. Disclosures Gordeuk: Emmaus Life Sciences: Consultancy.

Elevated Levels of Hgfl Protein in Sickle Cell Disease Urine Samples That Induce Glomerular Permeability

Background: Chronic kidney disease (CKD) is a prevalent complication of sickle cell disease (SCD) associated with early mortality. Hemoglobinuria is a risk factor for the development of albuminuria and CKD. Currently, there are no biomarkers that predict outcome of CKD. Mass-spectrometry analysis of patient urine is a highly potent modern method for biomarker discovery. An in vitro glomerular permeability assay has been used as a non-invasive test for glomerular disease prognosis. Application of this assay to urinary samples collected before kidney disease onset provides a unique opportunity for differential proteomics of a limited number of urinary proteins. In combination with high resolution/selected ion monitoring (HR/SIM) mass-spectrometry methodology, this experimental platform provides an opportunity for biomarker discovery and validation of proteins whose concentration is too low to be detected by an immunological assay. Objectives: We aimed to determine a biomarker of early stage kidney disease using samples collected from the patients of the Center for Sickle Cell Disease at Howard University. We then used HR/SIM method to validate this biomarker in the cohort of SCD patients with and without CKD from University of Illinois at Chicago (UIC). Methods:Urinary protein, creatinine, albumin were measured by ELISA. The pH and specific gravity (SG) were determined by Multistix. Glomeruli were isolated from the murine kidney (FVB/N strain) and albumin permeability (Palb) activity was determined using urine samples collected from patients of the Center for Sickle Cell Disease, Howard University. Mass-spectrometry analysis was performed and Protein Discovery 1.4 and SIEVE 2.0 programs were used for protein analysis and label-free quantification. Heavy isotope labeled peptide EDQTSPAPGLR(13C6, 15N) was used as an internal standard for HR/SIM analysis of the samples from UIC. Results: Glomerular permeability assay was performed using six urinary samples collected from patients of the Center for Sickle Cell Disease, Howard University. Mass-spectrometry assay was performed for all samples. Samples with similar values of protein, albumin, creatinine, pH and SG, but different Palb activity were compared by SIEVE 2.0. Higher levels of hepatocyte growth factor-like (HGFL) protein were observed in three samples that induced glomerular permeability compared to three samples that did not. Since our attempts to produce antibodies to HGFL peptide for an ELISA assay were unsuccessful, we developed a HR/SIM method to measure HGFL peptide in urine. HR/SIM was performed for eight urine samples from the UIC cohort by measuring the ratio of ion peaks of HGFL peptide (m/z 585.79) and internal standard (IS) (m/z 590.80) (Fig. 1A). HGFL levels were found to be significantly increased (1.72-fold, p=0.015) in the urine samples of two patients at risk of developing renal disease based on the presence of hemoglobinuria compared to six samples without hemoglobinuria (Fig. 1B). Conclusions: Combination of in vitro glomerular permeability assay with mass-spectrometry method of protein discovery and HR/SIM validation assay may be a useful platform for discovery of biomarker of early stage of CKD. Urinary level of HGFL may serve as a prognostic marker for development of CKD in SCD patients. A limitation of our study is the small number of samples used for validation. Acknowledgments: This work was supported by NIH Research Grants 1P50HL118006, 1R01HL125005 and 5G12MD007597. The content is solely the responsibility of the authors and does not necessarily represent the official view of NHLBI, NIMHD or NIH. Figure 1 HR/SIM analysis of HGFL peptide in human urine. (A)The ion peaks of HGFL peptide m/z 585.79 and internal standard (IS) m/z 590.80 were chosen for the high resolution/selective ion monitoring (HR/SIM) analysis using LTQ Orbitrap XL™ mass spectrometer. Extracted ion chromatograms (EICs) were based on a ±0.01 Da mass extraction window (MEW) centered on the theoretical m/z. (B) SCD patients at risk for developing renal disease based on the presence of hemoglobinuria showed significantly increased HGFL levels (1.72-fold, p=0.015). Figure 1. HR/SIM analysis of HGFL peptide in human urine. (A)The ion peaks of HGFL peptide m/z 585.79 and internal standard (IS) m/z 590.80 were chosen for the high resolution/selective ion monitoring (HR/SIM) analysis using LTQ Orbitrap XL™ mass spectrometer. Extracted ion chromatograms (EICs) were based on a ±0.01 Da mass extraction window (MEW) centered on the theoretical m/z. (B) SCD patients at risk for developing renal disease based on the presence of hemoglobinuria showed significantly increased HGFL levels (1.72-fold, p=0.015). Disclosures No relevant conflicts of interest to declare.