Development of the Double Cyclic Peptide Ligand for Antibody Purification and Protein Detection

Development of a peptide-based affinity matrix and detection reagent is important for biomedical research and the biopharmaceutical industry. In the present work, we designed and synthesized an immunoglobin G (IgG)-binding peptide ligand, Fc-III-4C. Fc-III-4C is composed of 15 residues, and the 4 cysteine residues form 2 disulfide bonds to generate a double cyclic structure. The binding affinity of the Fc-III-4C peptide toward human IgG was determined to be 2.45 nM (<i>K</i>_d), which is higher than that of IgG with Protein A/G (Pro-A/G). Importantly, the Fc-III-4C peptide displayed high affinity to various IgGs from different species. Fc-III-4C immobilized agarose beads exhibited high stability and reusability when compared with that of the Pro-A/G-immobilized beads. The conjugate of Fc-III-4C with FITC was demonstrated to be suitable for immunofluorescence detection of proteins expressed in cells. These results demonstrate that the Fc-III-4C peptide is a useful affinity ligand for antibody purification and as a protein detection reagent

Table_1_HMGN2 and Histone H1.2: potential targets of a novel probiotic mixture for seasonal allergic rhinitis.XLSX

BackgroundAllergic rhinitis (AR) is a common nasal inflammatory disorder that severely affects an individual's quality of life (QoL) and poses a heavy financial burden. In addition to routine treatments, probiotic intervention has emerged as a promising strategy for preventing and alleviating allergic diseases. The main objective of this study was to determine the effect of a novel multi-strain probiotic mixture on AR symptoms and investigate potential targets underlying the probiotic intervention.MethodsA randomized, double-blind, placebo-controlled clinical study was conducted on AR patients who were allergic to autumnal pollens (n = 31). Placebo or a novel probiotic mixture, composed of Lactobacillus rhamnosus (L. rhamnosus) HN001, L. acidophilus NCFM, Bifidobacterium lactis (B. lactis) Bi-07, L. paracasei LPC-37, and L. reuteri LE16, was administered after 2 months. The therapeutic efficacy was evaluated by a symptom assessment scale. Before and during the pollen season, blood samples were collected, and peripheral blood mononuclear cells (PBMCs) were isolated for further tandem mass tags (TMTs)-based quantitative proteomic analyses. Potential targets and underlying pathological pathways were explored using bioinformatics methods.ResultsDuring the pollen season, the rhinoconjunctivitis symptom score of participants who were administered probiotics (probiotic group, n = 15) was significantly lower than those administered placebo (placebo group, n = 15) (P = 0.037). The proteomic analyses identified 60 differentially expressed proteins (DEPs) in the placebo group, and subsequent enrichment analyses enriched a series of pathways and biological processes, including signaling pathways of inflammation, coagulation cascade, lipid, carbohydrate and amino acid metabolic pathways, and transcription and translation processes. Least Absolute Shrinkage and Selection Operator (LASSO) regression extracted five main elements, namely, GSTO1, ATP2A2, MCM7, PROS1, and TRIM58, as signature proteins. A total of 17 DEPs were identified in the probiotic group, and there was no pathway enriched. Comparison of DEPs in the two groups revealed that the expression levels of the high-mobility group nucleosome-binding domain-containing protein 2 (HMGN2) and Histone H1.2 presented an opposite trend with different interventions.ConclusionOur data showed that AR symptoms alleviated after treatment with the novel multi-strain probiotic mixture, and the proteomic analyses suggested that HMGN2 and Histone H1.2 might be targets of probiotic intervention for seasonal AR.</p

Table_3_HMGN2 and Histone H1.2: potential targets of a novel probiotic mixture for seasonal allergic rhinitis.XLS

BackgroundAllergic rhinitis (AR) is a common nasal inflammatory disorder that severely affects an individual's quality of life (QoL) and poses a heavy financial burden. In addition to routine treatments, probiotic intervention has emerged as a promising strategy for preventing and alleviating allergic diseases. The main objective of this study was to determine the effect of a novel multi-strain probiotic mixture on AR symptoms and investigate potential targets underlying the probiotic intervention.MethodsA randomized, double-blind, placebo-controlled clinical study was conducted on AR patients who were allergic to autumnal pollens (n = 31). Placebo or a novel probiotic mixture, composed of Lactobacillus rhamnosus (L. rhamnosus) HN001, L. acidophilus NCFM, Bifidobacterium lactis (B. lactis) Bi-07, L. paracasei LPC-37, and L. reuteri LE16, was administered after 2 months. The therapeutic efficacy was evaluated by a symptom assessment scale. Before and during the pollen season, blood samples were collected, and peripheral blood mononuclear cells (PBMCs) were isolated for further tandem mass tags (TMTs)-based quantitative proteomic analyses. Potential targets and underlying pathological pathways were explored using bioinformatics methods.ResultsDuring the pollen season, the rhinoconjunctivitis symptom score of participants who were administered probiotics (probiotic group, n = 15) was significantly lower than those administered placebo (placebo group, n = 15) (P = 0.037). The proteomic analyses identified 60 differentially expressed proteins (DEPs) in the placebo group, and subsequent enrichment analyses enriched a series of pathways and biological processes, including signaling pathways of inflammation, coagulation cascade, lipid, carbohydrate and amino acid metabolic pathways, and transcription and translation processes. Least Absolute Shrinkage and Selection Operator (LASSO) regression extracted five main elements, namely, GSTO1, ATP2A2, MCM7, PROS1, and TRIM58, as signature proteins. A total of 17 DEPs were identified in the probiotic group, and there was no pathway enriched. Comparison of DEPs in the two groups revealed that the expression levels of the high-mobility group nucleosome-binding domain-containing protein 2 (HMGN2) and Histone H1.2 presented an opposite trend with different interventions.ConclusionOur data showed that AR symptoms alleviated after treatment with the novel multi-strain probiotic mixture, and the proteomic analyses suggested that HMGN2 and Histone H1.2 might be targets of probiotic intervention for seasonal AR.</p

Table_4_HMGN2 and Histone H1.2: potential targets of a novel probiotic mixture for seasonal allergic rhinitis.XLS

BackgroundAllergic rhinitis (AR) is a common nasal inflammatory disorder that severely affects an individual's quality of life (QoL) and poses a heavy financial burden. In addition to routine treatments, probiotic intervention has emerged as a promising strategy for preventing and alleviating allergic diseases. The main objective of this study was to determine the effect of a novel multi-strain probiotic mixture on AR symptoms and investigate potential targets underlying the probiotic intervention.MethodsA randomized, double-blind, placebo-controlled clinical study was conducted on AR patients who were allergic to autumnal pollens (n = 31). Placebo or a novel probiotic mixture, composed of Lactobacillus rhamnosus (L. rhamnosus) HN001, L. acidophilus NCFM, Bifidobacterium lactis (B. lactis) Bi-07, L. paracasei LPC-37, and L. reuteri LE16, was administered after 2 months. The therapeutic efficacy was evaluated by a symptom assessment scale. Before and during the pollen season, blood samples were collected, and peripheral blood mononuclear cells (PBMCs) were isolated for further tandem mass tags (TMTs)-based quantitative proteomic analyses. Potential targets and underlying pathological pathways were explored using bioinformatics methods.ResultsDuring the pollen season, the rhinoconjunctivitis symptom score of participants who were administered probiotics (probiotic group, n = 15) was significantly lower than those administered placebo (placebo group, n = 15) (P = 0.037). The proteomic analyses identified 60 differentially expressed proteins (DEPs) in the placebo group, and subsequent enrichment analyses enriched a series of pathways and biological processes, including signaling pathways of inflammation, coagulation cascade, lipid, carbohydrate and amino acid metabolic pathways, and transcription and translation processes. Least Absolute Shrinkage and Selection Operator (LASSO) regression extracted five main elements, namely, GSTO1, ATP2A2, MCM7, PROS1, and TRIM58, as signature proteins. A total of 17 DEPs were identified in the probiotic group, and there was no pathway enriched. Comparison of DEPs in the two groups revealed that the expression levels of the high-mobility group nucleosome-binding domain-containing protein 2 (HMGN2) and Histone H1.2 presented an opposite trend with different interventions.ConclusionOur data showed that AR symptoms alleviated after treatment with the novel multi-strain probiotic mixture, and the proteomic analyses suggested that HMGN2 and Histone H1.2 might be targets of probiotic intervention for seasonal AR.</p

Table_2_HMGN2 and Histone H1.2: potential targets of a novel probiotic mixture for seasonal allergic rhinitis.XLSX

BackgroundAllergic rhinitis (AR) is a common nasal inflammatory disorder that severely affects an individual's quality of life (QoL) and poses a heavy financial burden. In addition to routine treatments, probiotic intervention has emerged as a promising strategy for preventing and alleviating allergic diseases. The main objective of this study was to determine the effect of a novel multi-strain probiotic mixture on AR symptoms and investigate potential targets underlying the probiotic intervention.MethodsA randomized, double-blind, placebo-controlled clinical study was conducted on AR patients who were allergic to autumnal pollens (n = 31). Placebo or a novel probiotic mixture, composed of Lactobacillus rhamnosus (L. rhamnosus) HN001, L. acidophilus NCFM, Bifidobacterium lactis (B. lactis) Bi-07, L. paracasei LPC-37, and L. reuteri LE16, was administered after 2 months. The therapeutic efficacy was evaluated by a symptom assessment scale. Before and during the pollen season, blood samples were collected, and peripheral blood mononuclear cells (PBMCs) were isolated for further tandem mass tags (TMTs)-based quantitative proteomic analyses. Potential targets and underlying pathological pathways were explored using bioinformatics methods.ResultsDuring the pollen season, the rhinoconjunctivitis symptom score of participants who were administered probiotics (probiotic group, n = 15) was significantly lower than those administered placebo (placebo group, n = 15) (P = 0.037). The proteomic analyses identified 60 differentially expressed proteins (DEPs) in the placebo group, and subsequent enrichment analyses enriched a series of pathways and biological processes, including signaling pathways of inflammation, coagulation cascade, lipid, carbohydrate and amino acid metabolic pathways, and transcription and translation processes. Least Absolute Shrinkage and Selection Operator (LASSO) regression extracted five main elements, namely, GSTO1, ATP2A2, MCM7, PROS1, and TRIM58, as signature proteins. A total of 17 DEPs were identified in the probiotic group, and there was no pathway enriched. Comparison of DEPs in the two groups revealed that the expression levels of the high-mobility group nucleosome-binding domain-containing protein 2 (HMGN2) and Histone H1.2 presented an opposite trend with different interventions.ConclusionOur data showed that AR symptoms alleviated after treatment with the novel multi-strain probiotic mixture, and the proteomic analyses suggested that HMGN2 and Histone H1.2 might be targets of probiotic intervention for seasonal AR.</p

Expansion of Endothelial Progenitor Cells in High Density Dot Culture of Rat Bone Marrow Cells

<div><p>In vitro expansion of endothelial progenitor cells (EPCs) remains a challenge in stem cell research and its application. We hypothesize that high density culture is able to expand EPCs from bone marrow by mimicking cell-cell interactions of the bone marrow niche. To test the hypothesis, rat bone marrow cells were either cultured in high density (2×10⁵ cells/cm²) by seeding total 9×10⁵ cells into six high density dots or cultured in regular density (1.6×10⁴ cells/cm²) with the same total number of cells. Flow cytometric analyses of the cells cultured for 15 days showed that high density cells exhibited smaller cell size and higher levels of marker expression related to EPCs when compared to regular density cultured cells. Functionally, these cells exhibited strong angiogenic potentials with better tubal formation in vitro and potent rescue of mouse ischemic limbs in vivo with their integration into neo-capillary structure. Global gene chip and ELISA analyses revealed up-regulated gene expression of adhesion molecules and enhanced protein release of pro-angiogenic growth factors in high density cultured cells. In summary, high density cell culture promotes expansion of bone marrow contained EPCs that are able to enhance tissue angiogenesis via paracrine growth factors and direct differentiation into endothelial cells.</p></div

Up-regulation of cell adhesion molecules and pro-angiogenic growth factors in high density cultured cells.

<p>A, Pathway analysis of microarray data. Genes involved in focal adhesion and ECM-receptor interactions were highly expressed in high density cultured cells compared with those in regular density cultured cells. B, Expression of integrin family genes validated by qRT-PCR analysis. Data are presented as the fold increase of gene expression in high density (HD) cultured cells compared with that in regular density (RD) cultured cells (n = 3). *p<0.05. C, Expression of growth factors validated by qRT-PCR analysis. Data are presented as the fold increase of gene expression in high density cultured cells compared with that in regular density cultured cells. (n = 3). *p<0.05. D, ELISA analysis of VEGF, PDGF, TGF-β, HGF, bFGF and SDF-1α in the supernatants of cultured cells. (n = 3). *p<0.05. E, Tube formation of HUVECs on Matrigel induced by conditioned medium from regular (CM-RD) or high density (CM-HD) cultures. Cells incubated in DMEM with 10% FBS served as a control medium (Ctrl-M). The number of branch points per field was counted after 12 hours of network formation (n = 9). *p<0.05. F, Growth factor expression of CD45⁺ and CD45⁻ cells sorted from high density culture validated by qRT-PCR analysis. (n = 3). *p<0.05.</p

Engraftment and endothelial differentiation of high density cultured cells in ischemic limbs.

<p>A and B, Confocal images revealed that some of high density cultured cells (CM-DiI⁺, white arrows) participated in neovascularization by direct differentiation into endothelial cells (ILB4⁺). C, CM-DiI⁺ cells (white arrows) close to capillaries, but negative for ILB4 staining, might support vessel formation via paracrine effect of released pro-angiogenic growth factors. D, The percentages of endothelial and non-endothelial differentiation of injected cells, and the percentage of vessels derived from donor and recipient (n = 3). *p<0.05.</p

Pro-angiogenic potential of high density cultured cells in vitro and in vivo.

<p>A, In vitro tube formation assay with Matrigel for the cells derived from high density (HD) and regular density (RD) groups with quantification of the number of branch points per field (n = 9). *p<0.05, Scale bars, 100 µm. B, An in vivo angiogenic assay was performed by transplantation of expanded cells into ischemic hind limbs of nude mice. Representative views of ischemic left hind limbs at 3 weeks after treatment with PBS, regular density cultured cells or high density cultured cells and percent distribution of outcomes after treatment (n = 10). C, Laser Doppler images of blood perfusion with mice in a supine position at day 1 after femoral artery ligation and at day 21 after cell transplantation or PBS injection. Restoration of blood flow was calculated by comparing the laser Doppler image data of ischemic left limbs with that of non-ischemic right limbs at day 21. A significant improvement of blood supply was achieved in the group treated with high density cultured cells (n = 4). *p<0.05. D, Blood vessels in hind limb adductor muscles were identified by ILB4 staining. A higher number of capillaries were observed in the group treated with high density cultured cells (n = 4). *p<0.05, Scale bars, 100 µm. E, Donor-derived cells were detected by CM-DiI labeling of hind limb adductor muscles. A higher number of CM-DiI⁺ cells were observed in the muscles treated with high density cultured cells (n = 5). *p<0.05, Scale bars, 100 µm.</p

Bone marrow cells in high density culture.

<p>A, Small bright cells were observed in high density culture (2×10⁵ cells/cm²) of rat bone marrow cells. Cells were incubated with DiI-ac-LDL and stained with FITC-conjugated UEA lectin and DAPI. Small bright cells were double-positive for DiI-ac-LDL and UEA lectin with counterstained DAPI. B, Spindle-shaped cells were observed in regular density culture (1.6×10⁴ cells/cm²). The majority of cells were negative for DiI-ac-LDL uptake and UEA lectin binding. C, Bone marrow cells were seeded at different densities. After 3 days of culture, fluorescence microscopic observation and flow cytometric analysis revealed an increase of DiI-ac-LDL-positive cells with the increase of cell seeding density (n = 3). Scale bars, 100 µm.</p