Quantitation of Acetyl Hexapeptide-8 in Cosmetics by Hydrophilic Interaction Liquid Chromatography Coupled to Photo Diode Array Detection

Bioactive peptides are gaining more and more popularity in the research and development of cosmetic products with anti-aging effect. Acetyl hexapeptide-8 is a hydrophilic peptide incorporated in cosmetics to reduce the under-eye wrinkles and the forehead furrows. Hydrophilic interaction liquid chromatography (HILIC) is the separation technique of choice for analyzing peptides. In this work, a rapid HILIC method coupled to photodiode array detection operated at 214 nm was developed, validated and used to determine acetyl-hexapeptide-8 in cosmetics. Chromatography was performed on a Xbridge® HILIC BEH analytical column using as mobile phase a 40 mM ammonium formate water solution (pH 6.5)-acetonitrile mixture 30:70, v/v at flow rate 0.25 mL min−1. The assay was linear over the concentration range 20 to 30 μg mL−1 for the cosmetic formulations and 0.004 to 0.007% (w/w) for the cosmetic cream. The limits of quantitation for acetyl hexapeptide-8 were 1.5 μg mL−1 and 0.002% (w/w) for the assay of cosmetic formulations and cosmetic creams, respectively. The method was applied to the analysis of cosmetic formulations and anti-wrinkle cosmetic creams.

A Germline-Encoded Structural Arginine Trap Underlies the Anti-DNA Reactivity of a Murine V Gene Segment

Autoimmunity may have its origins of early repertoire selection in developmental B cells. Such a primary repertoire is probably shaped by selecting B cells that can efficiently perform productive signaling, stimulated by self-antigens in the bone marrow, such as DNA. In support of that idea, we previously found a V segment from VH10 family that can form antibodies that bind to DNA independent of CDR3 usage. In this paper we designed four antibody fragments in a novel single-chain pre-BCR (scpre-BCR) format containing germinal V gene segments from families known to bind DNA (VH10) or not (VH4) connected to a murine surrogate light chain (SLC), lacking the highly charged unique region (UR), by a hydrophilic peptide linker. We also tested the influence of CDR2 on DNA reactivity by shuffling the CDR2 loop. The scpre-BCRs were expressed in bacteria. VH10 bearing scpre-BCR could bind DNA, while scpre-BCR carrying the VH4 segment did not. The CDR2 loop shuffling hampered VH10 reactivity while displaying a gain-of-function in the nonbinding VH4 germline. We modeled the binding sites demonstrating the conservation of a positivity charged pocket in the VH10 CDR2 as the possible cross-reactive structural element. We presented evidence of DNA reactivity hardwired in a V gene, suggesting a structural mechanism for innate autoreactivity. Therefore, while autoreactivity to DNA can lead to autoimmunity, efficiently signaling for B cell development is likely a trade-off mechanism leading to the selection of potentially autoreactive repertoires.

Lanthanide DO3A-Complexes Bearing Peptide Substrates: The Effect of Peptidic Side Chains on Metal Coordination and Relaxivity

Two DO3A-type ligands conjugated to substrates of urokinase (L3) and caspase-3 (L4) via a propyl-amide linker were synthesized and their lanthanide(III) (Ln3+) complexes studied. A model compound without peptide substrate (L2) and an amine derivative ligand mimicking the state after enzymatic cleavage (L1) were also prepared. Proton Nuclear Magnetic Relaxation Dispersion (NMRD) profiles recorded on the gadolinium(III) (Gd3+) complexes, complemented with the assessment of hydration numbers via luminescence lifetime measurements on the Eu3+ analogues, allowed us to characterize the lanthanide coordination sphere in the chelates. These data suggest that the potential donor groups of the peptide side chains (carboxylate, amine) interfere in metal coordination, leading to non-hydrated LnL3 and LnL4 complexes. Nevertheless, GdL3 and GdL4 retain a relatively high relaxivity due to an important second-sphere contribution generated by the strongly hydrophilic peptide chain. Weak PARACEST effects are detected for the amine-derivative EuL1 and NdL1 chelates. Unfortunately, the GdL3 and GdL4 complexes are not significantly converted by the enzymes. The lack of enzymatic recognition of these complexes can likely be explained by the participation of donor groups from the peptide side chain in metal coordination.

Charge-Mediated Co-assembly of Amphiphilic Peptide and Antibiotics Into Supramolecular Hydrogel With Antibacterial Activity

Bacteria are the most common pathogens to cause infection of surgical sites, which usually induce severe postoperative morbidity and more healthcare costs. Inhibition of bacteria adhesion and colonization is an effective strategy to prevent the spread of infection at the surgical sites. Hydrogels have been widely used as promising antibacterial materials, due to their unique porous structure that could accommodate various antibacterial agents (e.g., antibiotics and cationic polymers with inherent antibacterial activity). Herein, inspired by the natural protein self-assembly, an amphiphilic peptide comprised of a hydrophobic naphthyl (Nap) acetyl tail and a hydrophilic peptide backbone was employed to construct supramolecular hydrogel for sustained release of the antibiotic polymyxin B. At neutral pH, the negatively charged amphiphilic peptide could form electrostatic attraction interaction with the positively charged polymyxin B, which could thus drive the ionized peptide molecules to get close to each other and subsequently trigger the self-assembly of the amphiphilic peptide into supramolecular hydrogel via intermolecular hydrogen bonding interaction among the peptide backbones and π-stacking of the hydrophobic Nap tails. More importantly, the electrostatic attraction interaction between polymyxin B and the amphiphilic peptide could ensure the sustained release of polymyxin B from the supramolecular hydrogel, leading to an effective inhibition of Gram-negative bacteria Escherichia coli growth. Combining the good biocompatibility of the amphiphilic peptide, the supramolecular hydrogel developed in this work shows a great potential for the surgical site infection application.

Peptidome analysis of cerebrospinal fluid in neonates with hypoxic-ischemic brain damage

Hypoxic-ischemic brain injury (HIBD) causes neonatal death and serious neurological disability; however, there are currently no promising therapies for it excepting cooling. Therefore, in this study, we used peptidome analysis to identify differentially expressed peptides in cerebrospinal fluid (CSF) of neonates with HIBD or controls, which may give a foundation for finding new promising drugs of neonatal HIBD. CSF samples were collected from neonates with HIBD (n = 4) or controls (n = 4). ITRAQ LC–MS/MS was used to identify differentially expressed peptides between two groups. A total of 35 differentially expressed peptides from 25 precursor proteins were identified. The 2671.5 Da peptide (HSQFIGYPITLFVEKER), one of the down-regulated peptides in neonatal HIBD, is a fragment of heat shock protein 90-alpha (HSP90α/HSP90AA1). Results of bioinformatics analysis showed that HSP90α/HSP90AA1 was located in the protein–protein interaction (PPI) network hub and was involved in the NOD-LIKE receptor (NLR) signaling pathway. This peptide, we named it Hypoxic-Ischemic Brain Damage Associated Peptide (HIBDAP), is a hydrophilic peptide with high stability and has a long half-life of 3.5 h in mammalian reticulocytes. It was demonstrated that TAT-HIBDAP could successfully enter PC12 cells and further into the nucleus. After HIBDAP pretreatment and 6 h of OGD treatment, low concentrations of HIBDAP increased the survival rate of cells, except 40 μM had a toxic effect. Safe concentrations of HIBDAP reduced pyroptosis of PC12 cells under OGD, except 20 μM had no effect, by suppressing expressions of NLRP3, ASC and Caspase-1 except NLRP1. The results of our study identified the characterization and expression profiles of peptides in CSF of neonatal HIBD. Several meaningful peptides such as HIBDAP may play significant roles in neonatal HIBD and provide new therapeutic targets for neonatal HIBD.

Peptidome analysis of cerebrospinal fluid in neonates with hypoxic-ischemic brain damage

Hypoxic-ischemic brain injury (HIBD) causes neonatal death and serious neurological disability; however, there are currently no promising therapies for it excepting cooling. Therefore, in this study, we used peptidome analysis to identify differentially expressed peptides in cerebrospinal fluid (CSF) of neonates with HIBD or controls, which may give a foundation for finding new promising drugs of neonatal HIBD. CSF samples were collected from neonates with HIBD (n=4) or controls (n=4). ITRAQ LC-MS/MS was used to identify differentially expressed peptides between two groups. A total of 35 differentially expressed peptides from 25 precursor proteins were identified. The 2671.5Da peptide (HSQFIGYPITLFVEKER), one of the down-regulated peptides in neonatal HIBD, is a fragment of heat shock protein 90-alpha (HSP90α/HSP90AA1). Results of bioinformatics analysis showed that HSP90α/HSP90AA1 was located in the protein-protein interaction (PPI) network hub and was involved in the NOD-LIKE receptor (NLR) signaling pathway. This peptide, we named it HIBDAP (Hypoxic-Ischemic Brain Damage Associated Peptide), is a hydrophilic peptide with high stability and has a long half-life of 3.5h in mammalian reticulocytes. It was demonstrated that TAT-HIBDAP could successfully enter PC12 cells and further into the nucleus. After HIBDAP pretreatment and 6 hours of OGD treatment, low concentrations of HIBDAP increased the survival rate of cells, except 40μM had a toxic effect. Safe concentrations of HIBDAP reduced pyroptosis of PC12 cells under OGD, except 20μM had no effect, by suppressing expressions of NLRP3, ASC and Caspase-1 except NLRP1. The results of our study identified the characterization and expression profiles of peptides in CSF of neonatal HIBD. Several meaningful peptides such as HIBDAP may play significant roles in neonatal HIBD and provide new therapeutic targets for neonatal HIBD.

Fiber Crimp Confers Matrix Mechanical Nonlinearity, Regulates Endothelial Cell Mechanosensing, and Promotes Microvascular Network Formation

Mechanical interactions between cells and their surrounding extracellular matrix (ECM) guide many fundamental cell behaviors. Native connective tissue consists of highly organized, 3D networks of ECM fibers with complex, nonlinear mechanical properties. The most abundant stromal matrix component is fibrillar type I collagen, which often possesses a wavy, crimped morphology that confers strain- and load-dependent nonlinear mechanical behavior. Here, we established a new and simple method for engineering electrospun fibrous matrices composed of dextran vinyl sulfone (DexVS) with controllable crimped structure. A hydrophilic peptide was functionalized to DexVS matrices to trigger swelling of individual hydrogel fibers, resulting in crimped microstructure due to the fixed anchorage of fibers. Mechanical characterization of these matrices under tension confirmed orthogonal control over nonlinear stress–strain responses and matrix stiffness. We next examined ECM mechanosensing of individual endothelial cells (ECs) and found that fiber crimp promoted physical matrix remodeling alongside decreases in cell spreading, focal adhesion area, and nuclear localization of Yes-associated protein (YAP). These changes corresponded to an increase in migration speed, along with evidence for long-range interactions between neighboring cells in crimped matrices. Interestingly, when ECs were seeded at high density in crimped matrices, capillary-like networks rapidly assembled and contained tube-like cellular structures wrapped around bundles of synthetic matrix fibers due to increased physical reorganization of matrix fibers. Our work provides an additional level of mechanical and architectural tunability to synthetic fibrous matrices and implicates a critical role for mechanical nonlinearity in EC mechanosensing and network formation.

M13 phage display to identify a permeating peptide against hyperconcentrated mucin

ABSTRACTMucus is an impregnable barrier for drug delivery across the epithelia for treatment of mucosal-associated diseases. While current carriers are promising for mucus penetration, their surface chemistries do not possess chemical complexity to probe and identify optimal physicochemical properties desired for mucus penetration. As initial study, we use M13 phage display presenting random peptides to select peptides that can facilitate permeation through hyperconcentrated mucin. Here, a net-neutral charge, hydrophilic peptide was identified to facilitate transport of phage and fluorophore conjugates through mucin barrier compared to controls. This initial finding warrants further study to understand how composition and spatial distribution of physicochemical properties of peptides can be optimized to improve transport across the mucus barrier.