Synthesis of Caffeoyl-Prolyl-Histidyl-Xaa Derivatives and Evaluation of Their Activities and Stability upon Long-Term Storage.

Antioxidants play a critical role in the treatment of degenerative diseases and delaying the aging of dermal tissue. Caffeic acid (CA) is a representative example of the antioxidants found in plants. However, CA is unsuitable for long-term storage because of its poor stability under ambient conditions. Caffeoyl-Pro-His-NH2 (CA-Pro-His-NH2, CA-PH) exhibits the highest antioxidant activity, free radical scavenging and lipid peroxidation inhibition activity among the histidine-containing CA-conjugated dipeptides reported to date. The addition of short peptides to CA, such as Pro-His, is assumed to synergistically enhance its antioxidative activity. In this study, several caffeoyl-prolyl-histidyl-Xaa-NH2 derivatives were synthesized and their antioxidative activities evaluated. CA-Pro-His-Asn-NH2 showed enhanced antioxidative activity and higher structural stability than CA-PH, even after long-term storage. CA-Pro-His-Asn-NH2 was stable for 3 months, its stability being evaluated by observing the changes in its NMR spectra. Moreover, the solid-phase synthetic strategy used to prepare these CA-Pro-His-Xaa-NH2 derivatives was optimized for large-scale production. We envision that CA-Pro-His-Xaa-NH2 derivatives can be used as potent dermal therapeutic agents and useful cosmetic ingredients.

Robust Magnetized Graphene Oxide Platform for In Situ Peptide Synthesis and FRET-Based Protease Detection.

Graphene oxide (GO)/peptide complexes as a promising disease biomarker analysis platform have been used to detect proteolytic activity by observing the turn-on signal of the quenched fluorescence upon the release of peptide fragments. However, the purification steps are often cumbersome during surface modification of nano-/micro-sized GO. In addition, it is still challenging to incorporate the specific peptides into GO with proper orientation using conventional immobilization methods based on pre-synthesized peptides. Here, we demonstrate a robust magnetic GO (MGO) fluorescence resonance energy transfer (FRET) platform based on in situ sequence-specific peptide synthesis of MGO. The magnetization of GO was achieved by co-precipitation of an iron precursor solution. Magnetic purification/isolation enabled efficient incorporation of amino-polyethylene glycol spacers and subsequent solid-phase peptide synthesis of MGO to ensure the oriented immobilization of the peptide, which was evaluated by mass spectrometry after photocleavage. The FRET peptide MGO responded to proteases such as trypsin, thrombin, and ß-secretase in a concentration-dependent manner. Particularly, ß-secretase, as an important Alzheimer's disease marker, was assayed down to 0.125 ng/mL. Overall, the MGO platform is applicable to the detection of other proteases by using various peptide substrates, with a potential to be used in an automated synthesis system operating in a high throughput configuration.

Caffeoyl-Prolyl-Histidine Amide Inhibits Fyn and Alleviates Atopic Dermatitis-Like Phenotypes via Suppression of NF-κB Activation.

Caffeic acid (CA) is produced from a variety of plants and has diverse biological functions, including anti-inflammation activity. It has been recently demonstrated that caffeoyl-prolyl-histidine amide (CA-PH), which is CA conjugated with proline-histidine dipeptide, relieves atopic dermatitis (AD)-like phenotypes in mouse. In this study, we investigated the molecular mechanism underlying CA-PH-mediated alleviation of AD-like phenotypes using cell line and AD mouse models. We confirmed that CA-PH suppresses AD-like phenotypes, such as increased epidermal thickening, infiltration of mast cells, and dysregulated gene expression of cytokines. CA-PH suppressed up-regulation of cytokine expression through inhibition of nuclear translocation of NF-κB. Using a CA-PH affinity pull-down assay, we found that CA-PH binds to Fyn. In silico molecular docking and enzyme kinetic studies revealed that CA-PH binds to the ATP binding site and inhibits Fyn competitively with ATP. CA-PH further suppressed spleen tyrosine kinase (SYK)/inhibitor of nuclear factor kappa B kinase (IKK)/inhibitor of nuclear factor kappa B (IκB) signaling, which is required for nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation. In addition, chronic application of CA-PH, in contrast with that of glucocorticoids, did not induce up-regulation of regulated in development and DNA damage response 1 (REDD1), reduction of mammalian target of rapamycin (mTOR) signaling, or skin atrophy. Thus, our study suggests that CA-PH treatment may help to reduce skin inflammation via down-regulation of NF-κB activation, and Fyn may be a new therapeutic target of inflammatory skin diseases, such as AD.

Preparation of tri(ethylene glycol) grafted core-shell type polymer support for solid-phase peptide synthesis.

A core-shell type polymer support for solid-phase peptide synthesis has been developed for high coupling efficiency of peptides and versatile applications such as on-bead bioassays. Although various kinds of polymer supports have been developed, they have their own drawbacks including poor accessibility of reagents and incompatibility in aqueous solution. In this paper, we prepared hydrophilic tri(ethylene glycol) (TEG) grafted core-shell type polymer supports (TEG SURE) for efficient solid-phase peptide synthesis and on-bead bioassays. TEG SURE was prepared by grafting TEG derivative on the surface of AM PS resin via biphasic diffusion control method and subsequent acetylation of amine groups which are located at the core region of AM PS resin. The performance of TEG SURE was evaluated by synthesizing several peptides. Three points can be highlighted: (1) easy control of loading level of TEG, (2) improved efficiency of peptide synthesis compared with the conventional resins, and (3) applicability of on-bead bioassays.

Development of an aptasensor for electrochemical detection of exosomes.

Exosomes are small (50-100 nm in diameter) vesicles secreted from various mammalian cells. Exosomes have been correlated with tumor antigens and anti-tumor immune responses and may represent cancer biomarkers. Herein, we report on the development of an aptamer-based electrochemical biosensor for quantitative detection of exosomes. Aptamers specific to exosome transmembrane protein CD63 were immobilized onto gold electrode surfaces and incorporated into a microfluidic system. Probing strands pre-labeled with redox moieties were hybridized onto aptamer molecules anchored on the electrode surface. In the presence of exosomes these beacons released probing strands with redox reporters causing electrochemical signal to decrease. These biosensors could be used to detect as few as 1×10(6) particles/mL of exosomes, which represents 100-fold decrease in the limit of detection compared to commercial immunoassays relying on anti-CD63 antibodies. Given the importance of exosome-mediated signal transmission among cells, our study may represent an important step towards development of a simple biosensor that detects exosomes without washing or labeling steps in complex media.

Microfluidic compartments with sensing microbeads for dynamic monitoring of cytokine and exosome release from single cells.

Monitoring activity of single cells has high significance for basic science and diagnostic applications. Here we describe a reconfigurable microfluidic device for confining single cells along with antibody-modified sensing beads inside 20 picoliter (pL) microcompartments for monitoring cellular secretory activity. An array of ∼7000 microchambers fabricated in the roof of the reconfigurable microfluidic device could be raised or lowered by applying negative pressure. The floor of the device was micropatterned to contain cell attachment sites in registration with the microcompartments. Using this set-up, we demonstrated the detection of inflammatory cytokine IFN-γ and exosomes from single immune cells and cancer cells respectively. The detection scheme was similar in both cases: cells were first captured on the surface inside the microfluidic device, then sensing microbeads were introduced into the device so that, once the microcompartments were lowered, single cells and microbeads became confined together. The liquid bathing the beads and the cells inside the compartments also contained fluorescently-labeled secondary antibodies (Abs). The capture of cell-secreted molecules onto microbeads was followed by binding of secondary antibodies - this caused microbeads to become fluorescent. The fluorescence intensity of the microbeads changed over time, providing dynamics of single cell secretory activity. The microdevice described here may be particularly useful in the cases where panning upstream of sensing is required or to analyze secretory activity of anchorage-dependent cells.

Photodegradable hydrogels for capture, detection, and release of live cells.

Cells may be captured and released using a photodegradable hydrogel (photogel) functionalized with antibodies. Photogel substrates were used to first isolate human CD4 or CD8 T-cells from a heterogeneous cell suspension and then to release desired cells or groups of cells by UV-induced photodegradation. Flow cytometry analysis of the retrieved cells revealed approximately 95% purity of CD4 and CD8 T-cells, suggesting that this substrate had excellent specificity. To demonstrate the possibility of sorting cells according to their function, photogel substrates that were functionalized with anti-CD4 and anti-TNF-α antibodies were prepared. Single cells captured and stimulated on such substrates were identified by the fluorescence "halo" after immunofluorescent staining and could be retrieved by site-specific exposure to UV light through a microscope objective. Overall, it was demonstrated that functional photodegradable hydrogels enable the capture, analysis, and sorting of live cells.

Micropatterned sensing hydrogels integrated with reconfigurable microfluidics for detecting protease release from cells.

Matrix metalloproteinases (MMPs) play a central role in the breakdown of the extracellular matrix and are typically upregulated in cancer cells. The goal of the present study is to develop microwells suitable for capture of cells and detection of cell-secreted proteases. Hydrogel microwells comprised of poly(ethylene glycol) (PEG) were photopatterned on glass and modified with ligands to promote cell adhesion. To sense protease release, peptides cleavable by MMP9 were designed to contain a donor/acceptor FRET pair (FITC and DABCYL). These sensing molecules were incorporated into the walls of the hydrogel wells to enable a detection scheme where cells captured within the wells secreted protease molecules which diffused into the gel, cleaved the peptide, and caused a fluorescence signal to come on. By challenging sensing hydrogel microstructures to known concentrations of recombinant MMP9, the limit of detection was determined to be 0.625 nM with a linear range extending to 40 nM. To enhance sensitivity and to limit cross-talk between adjacent sensing sites, microwell arrays containing small groups (∼20 cells/well) of lymphoma cells were integrated into reconfigurable PDMS microfluidic devices. Using this combination of sensing hydrogel microwells and reconfigurable microfluidics, detection of MMP9 release from as few as 11 cells was demonstrated. Smart hydrogel microstructures capable of sequestering small groups of cells and sensing cell function have multiple applications ranging from diagnostics to cell/tissue engineering. Further development of this technology will include single-cell analysis and function-based cell sorting capabilities.

Micropatterned surfaces functionalized with electroactive peptides for detecting protease release from cells.

Matrix metalloproteinases (MMPs) regulate composition of the extracellular matrix and play a critical role in cancer, fibrosis, and wound healing. This article describes a novel peptide-based electrochemical biosensor for detecting activity of cell-secreted protease MMP9. In this sensing strategy, a peptide specific to MMP9 was modified with a redox label (methylene blue (MB)) and immobilized on microfabricated 300 µm diameter Au electrodes. Challenging the electrodes with known concentrations of MMP9 resulted in the cleavage of the MB containing peptide fragment and caused a decrease in electrical signal measured by square wave voltammetry (SWV). The limit of detection for MMP9 was determined to be 60 pM with a linear range extending to 50 nM. In preparation to detect cell-secreted MMP9, glass surfaces with Au electrode arrays were further micropatterned with poly(ethylene glycol) (PEG) gel to define annular cell adhesive regions next to electrodes and render the remainder of the surface nonfouling. The surfaces were further modified with CD14 antibody to promote attachment of monocytes. The peptide-modified electrode arrays were integrated into PDMS microfluidic devices and incubated with U-937 cells, transformed monocytes known to produce MMPs. These studies revealed a 3-fold higher electrochemical signal from ∼400 activated monocytes after 10 min activation compared to nonactivated monocytes. Whereas this article focuses on MMP9 detection, the general strategy of employing redox-labeled peptides on electrodes should be broadly applicable for detection of other proteases and should have clinical as well as basic science applications.

Aptamer-containing surfaces for selective capture of CD4 expressing cells.

Aptamers have recently emerged as an excellent alternative to antibodies because of their inherent stability and ease of modification. In this paper, we describe the development of an aptamer-based surface for capture of cells expressing CD4 antigen. The glass or silicon surfaces were modified with amine-terminated silanes and then modified with thiolated RNA aptamer against CD4. Modification of the surface was first characterized by ellipsometry to demonstrate assembly of biointerface components and to show specific capture of recombinant CD4 protein. Subsequently, surfaces were challenged with model lymphocytes (cell lines) that were either positive or negative for CD4 antigen. Our experiments show that aptamer-functionalized surfaces have similar capture efficiency to substrates containing anti-CD4 antibody. To mimick capture of specific T-cells from a complex cell mixture, aptamer-modified surfaces were exposed to binary mixtures containing Molt-3 cells (CD4+) spiked into Daudi B cells (CD4-). 94% purity of CD4 cells was observed on aptamer-containing surfaces from an initial fraction of 15% of CD4. Given the importance of CD4 cell enumeration in HIV/AIDS diagnosis and monitoring, aptamer-based devices may offer an opportunity for novel cell detection strategies and may yield more robust and less expensive blood analysis devices in the future.

Determination of protease subsite preference on SPOT peptide array by fluorescence quenching-based assay.

A peptide SPOT array was synthesized on a glass chip and used to determine protease subsite preference. To synthesize a peptide array for positional scanning, the ratio of the isokinetic concentration was determined for every Fmoc-amino acid except Cys. Based on this ratio, a peptide array consisting of Dabcyl-X-X-P(2)-Arg-X-X-X-Lys(FITC) (X: equimolar mixture of 19 amino acids, P(2): one of 19 amino acids) was synthesized on a chitosan-grafted glass chip. Subsequently, the peptide substrates on the array were hydrolyzed by thrombin to screen for subsite specificity using a fluorescence quenching-based assay. The P(2) subsite specificity of thrombin was screened by the fluorescence images obtained after hydrolysis. Pro at the P(2) subsite showed the highest specificity for thrombin based on both the fluorescence quenching-based assay and the solution phase assay. From these results, we confirmed that our mixture-based peptide SPOT array format on the chitosan-grafted glass chips could be used to determine protease subsite preference.

Electrochemical Impedance-Based Biosensors for the Label-Free Detection of the Nucleocapsid Protein from SARS-CoV-2.

Diagnosis of coronavirus disease (COVID-19) is important because of the emergence and global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Real-time polymerase chain reaction (PCR) is widely used to diagnose COVID-19, but it is time-consuming and requires sending samples to test centers. Thus, the need to detect antigens for rapid on-site diagnosis rather than PCR is increasing. We quantified the nucleocapsid (N) protein in SARS-CoV-2 using an electro-immunosorbent assay (El-ISA) and a multichannel impedance analyzer with a 96-interdigitated microelectrode sensor (ToAD). The El-ISA measures impedance signals from residual detection antibodies after sandwich assays and thus offers highly specific, label-free detection of the N protein with low cross-reactivity. The ToAD sensor enables the real-time electrochemical detection of multiple samples in conventional 96-well plates. The limit of detection for the N protein was 0.1 ng/mL with a detection range up to 10 ng/mL. This system did not detect signals for the S protein. While this study focused on detecting the N protein in SARS-CoV-2, our system can also be widely applicable to detecting various biomolecules involved in antigen-antibody interactions.

Antibody-free peptide substrate screening of serine/threonine kinase (protein kinase A) with a biotinylated detection probe.

Being different from anti-phosphotyrosine antibodies, anti-phosphoserine- or anti-phosphothreonine-specific antibodies with high affinity for the detection of serine/threonine kinase substrates are not readily available. Therefore, chemical modification methods were developed for the detection of phosphoserine or threonine in the screening of protein kinase substrates based on ß-elimination and Michael addition. We have developed a biotin-based detection probe for identification of the phosphorylated serine or threonine residue. A biotin derivative induced a color reaction using alkaline phosphate-conjugated streptavidin that amplified the signal. It was effective for the detection and separation of the target peptide on the resin. The detection probe was successfully used in identifying PKA substrates from peptide libraries on resin beads. The peptide library was prepared as a ladder-type, such that the active peptides on the colored resin beads were readily sequenced with the truncated peptide fragments by MALDI-TOF/MS analysis after releasing the peptides from the resin bead through photolysis.

Development of high-throughput phosphorylation profiling method for identification of Ser/Thr kinase specificity.

Identification of substrate specificity of kinases is crucial to understand the roles of the kinases in cellular signal transduction pathways. Here, we present an approach applicable for the discovery of substrate specificity of Ser/Thr kinases. The method, which is named as the 'high-throughput phosphorylation profiling (HTPP)' method was developed on the basis of a fully randomized one-bead one-compound (OBOC) combinatorial ladder type peptide library and MALDI-TOF MS. The OBOC ladder peptide library was constructed by the 'split and pool' method on a HiCore resin. The peptide library sequence was Ac-Ala-X-X-X-Ser-X-X-Ala-BEBE-PLL resin. The substrate specificity of murine PKA (cAMP-dependent protein kinase A) and yeast Yak1 kinase was identified using this method. On the basis of the result, we identified Ifh1, which is a co-activator for the transcription of ribosomal protein genes, as a novel substrate of Yak1 kinase. The putative Yak1-dependent phosphorylation site of Ifh1 was verified by in vitro kinase assay.

Silicone Implants Immobilized with Interleukin-4 Promote the M2 Polarization of Macrophages and Inhibit the Formation of Fibrous Capsules.

Breast augmentations with silicone implants can have adverse effects on tissues that, in turn, lead to capsular contracture (CC). One of the potential ways of overcoming CC is to control the implant/host interaction using immunomodulatory agents. Recently, a high ratio of anti-inflammatory (M2) macrophages to pro-inflammatory (M1) macrophages has been reported to be an effective tissue regeneration approach at the implant site. In this study, a biofunctionalized implant was coated with interleukin (IL)-4 to inhibit an adverse immune reaction and promoted tissue regeneration by promoting polarization of macrophages into the M2 pro-healing phenotype in the long term. Surface wettability, nitrogen content, and atomic force microscopy data clearly showed the successful immobilization of IL-4 on the silicone implant. Furthermore, in vitro results revealed that IL-4-coated implants were able to decrease the secretion of inflammatory cytokines (IL-6 and tumor necrosis factor-α) and induced the production of IL-10 and the upregulation of arginase-1 (mannose receptor expressed by M2 macrophage). The efficacy of this immunomodulatory implant was further demonstrated in an in vivo rat model. The animal study showed that the presence of IL-4 diminished the capsule thickness, the amount of collagen, tissue inflammation, and the infiltration of fibroblasts and myofibroblasts. These results suggest that macrophage phenotype modulation can effectively reduce inflammation and fibrous CC on a silicone implant conjugated with IL-4.

Automated maskless photolithography system for peptide microarray synthesis on a chip.

Maskless photolithographic peptide synthesis was performed on a glass chip using an automated peptide array synthesizer system. The peptide array synthesizer was built in a closed box, which contained optical and fluidic systems. The conditions for peptide synthesis were fully controlled by a computer program. For the peptide synthesis on a glass chip, 20 NVOC-protected amino acids were synthesized. The coupling efficiencies of two model peptide sequences were examined on ACA/APTS and PEG/CHI/GPTS chips. PEG/CHI/GPTS chip gave higher average stepwise yields of GIYWHHY (94%) and YIYGSFK (98%) than those of ACA/APTS chip. To quantify peptide-protein binding affinity, HPQ- or HPM-containing pentapeptides were synthesized on a PEG/CHI/GPTS chip and the binding event of Cy3 labeled-streptavidin was quantified. The peptide sequence of IQHPQ showed highest binding affinity with Cy3 labeled-streptavidin. The results demonstrated that the photolithographic peptide array synthesis method efficiently quantified the binding activities of protein-peptide interactions and it can be used for additional biological assay applications.

Two-Step Enzymatic Modification of Solid-Supported Bergenin in Aqueous and Organic Media.

The natural flavonoid bergenin was directly immobilized onto carboxylic acid functionalized controlled pore glass (carboxy-CPG) at 95% yield. Immobilized bergenin was brominated via chloroperoxidase in aqueous solution and then transesterified with vinyl butyrate in diisopropyl ether by subtilisin carslberg (SC) extracted into the organic solvent via ion pairing. Enzymatic cleavage of 7-bromo-4-butyrylbergenin from carboxy-CPG (9.6% final yield) was accomplished using lipase B (LipB) in an aqueous/organic mixture (90/10 v/v of water/acetonitrile), demonstrating the feasibility of solid phase biocatalysis of a natural product in aqueous and non-aqueous media.

High-throughput screening of glycan-binding proteins using miniature pig kidney N-glycan-immobilized beads.

Glycan recognition leading to cell-cell interactions, signaling, and immune responses is mediated by various glycan-binding proteins (GBPs) showing highly diverse ligand specificities. We describe here a rapid glycan immobilization technique via 4-hydrazinobenzoic acid (HBA)-functionalized beads and its application to high-throughput screening of miniature pig kidney N-glycan-binding proteins by using a mass-spectrometric approach. Without any derivatization steps, the purified pig kidney N-glycans were directly immobilized on to HBA-functionalized beads and subsequently used to identify GBPs from human serum. This screening method showed remarkable performance for identifying potential GBPs closely involved in pig-to-human xenograft rejection mediated by human serum, including antibodies, cytokines, complement components, siglec, and CD antigens. Thus, these results demonstrate that the GBP screening method was firmly established by one-step immobilization of the N-glycans on to microsphere and highly sensitive mass-spectrometric analysis.

Microfluidic Generation of Amino-Functionalized Hydrogel Microbeads Capable of On-Bead Bioassay.

Microfluidic generation of hydrogel microbeads is a highly efficient and reproducible approach to create various functional hydrogel beads. Here, we report a method to prepare crosslinked amino-functionalized polyethylene glycol (PEG) microbeads using a microfluidic channel. The microbeads generated from a microfluidic device were evaluated by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS) and confocal laser scanning microscopy, respectively. We found that the microbeads were monodisperse and the amino groups were localized on the shell region of the microbeads. A swelling test exhibited compatibility with various solvents. A cell binding assay was successfully performed with RGD peptide-coupled amino-functionalized hydrogel microbeads. This strategy will enable the large production of the various functional microbeads, which can be used for solid phase peptide synthesis and on-bead bioassays.