Rapid Endolysosomal Escape and Controlled Intracellular Trafficking of Cell Surface Mimetic Quantum-Dots-Anchored Peptides and Glycopeptides

A novel strategy for the development of a high performance nanoparticules platform was established by means of cell surface mimetic quantum-dots (QDs)-anchored peptides/glycopeptides, which was developed as a model system for nanoparticle-based drug delivery (NDD) vehicles with defined functions helping the specific intracellular trafficking after initial endocytosis. In this paper, we proposed a standardized protocol for the preparation of multifunctional QDs that allows for efficient cellular uptake and rapid escaping from the endolysosomal system and subsequent cytoplasmic molecular delivery to the target cellular compartment. Chemoselective ligation of the ketone-functionalized hexahistidine derivative facilitated both efficient endocytic entry and rapid endolysosomal escape of the aminooxy/phosphorylcholine self-assembled monolayer-coated QDs (AO/PCSAM-QDs) to the cytosol in various cell lines such as human normal and cancer cells, while modifications of these QDs with cell-penetrating arginine-rich peptides showed poor cellular uptake and induced self-aggregation of AO/PCSAM-QDs. Combined use of hexahistidylated AO/PCSAM-QDs with serglycine-like glycopeptides, namely synthetic proteoglycan initiators (PGIs), elicited the entry and controlled intracellular trafficking, Golgi localization, and also excretion of these nanoparticles, which suggested that the present approach would provide an ideal platform for the design of high performance NDD systems

Large-Scale Glycomics of Livestock: Discovery of Highly Sensitive Serum Biomarkers Indicating an Environmental Stress Affecting Immune Responses and Productivity of Holstein Dairy Cows

Because various stresses strongly influence the food productivity of livestock, biomarkers to indicate unmeasurable environmental stress in domestic animals are of increasing importance. Thermal comfort is one of the basic principles of dairy cow welfare that enhances productivity. To discover sensitive biomarkers that monitor such environmental stresses in dairy cows, we herein performed, for the first time, large-scale glycomics on 336 lactating Holstein cow serum samples over 9 months between February and October. Glycoblotting combined with MALDI-TOF/MS and DMB/HPLC allowed for comprehensive glycomics of whole serum glycoproteins. The results obtained revealed seasonal alterations in serum <i>N</i>-glycan levels and their structural characteristics, such as an increase in high-mannose type <i>N</i>-glycans in spring, the occurrence of di/triantennary complex type <i>N</i>-glycans terminating with two or three Neu5Gc residues in summer and autumn, and <i>N</i>-glycans in winter dominantly displaying Neu5Ac. A multivariate analysis revealed a correlation between the serum expression levels of these season-specific glycoforms and productivity

Serum Glycan Markers for Evaluation of Disease Activity and Prediction of Clinical Course in Patients with Ulcerative Colitis

<div><p>Background</p><p>The aims of this study were to determine the change of whole-serum <i>N</i>-glycan profile in ulcerative colitis (UC) patients and to investigate its clinical utility.</p><p>Methods</p><p>We collected serum from 75 UC patients at the time of admission and the same number of age/sex-matched healthy volunteers. Serum glycan profile was measured by comprehensive quantitative high-throughput glycome analysis and was compared with disease activity and prognosis.</p><p>Results</p><p>Out of 61 glycans detected, 24 were differentially expressed in UC patients. Pathway analysis demonstrated that highly sialylated multi-branched glycans and agalactosyl bi-antennary glycans were elevated in UC patients; in addition, the glycan ratio <i>m/z</i> 2378/1914, which also increased in UC, showed the highest Area under Receiver Operating Characteristic curve (0.923) for the diagnosis of UC. Highly sialylated multi-branched glycans and the glycan ratio <i>m/z</i> 2378/1914 were higher in the patients with total colitis, Clinical Activity Index >10, Mayo endoscopic score 3, or a steroid-refractory status. In particular, the glycan ratio <i>m/z</i> 2378/1914 (above median) was an independent prognostic factor for the need for an operation (hazard ratio, 2.67; 95% confidence interval, 1.04–7.84).</p><p>Conclusions</p><p>Whole-serum glycan profiles revealed that the glycan ratio <i>m/z</i> 2378/1914 and highly sialylated multi-branched glycans increase in UC patients, and are correlated with disease activity. The glycan ratio <i>m/z</i> 2378/1914 was an independent predictive factor of the prognosis of UC.</p></div

Pathway analysis of glycan biosynthesis.

<p>Relative glycan expression is indicated by <i>circle color</i> according to log2(patient/HLT) value. Color mapping is shown in the color bar. The <i>four-digit</i> numbers indicate the numbers of hexose (galactose and mannose), N-acetyl- hexosamine (N-acetyl glucosamine), fucose, and sialic acid moieties, in this order. All the samples used in this analysis were obtained from mean values of patients or healthy controls. Increased biosynthesis of multi-branched glycans, especially highly sialylated (≥3) glycans, and agalactosyl bi-antennary glycans was clearly observed in ulcerative colitis patients. Glycans <i>m/z</i> 2378 and 1914 correspond to 5402 and 5410 in this figure, respectively.</p

Prognostic factors for requiring an operation in patients with ulcerative colitis.

<p>Note: Only variables that demonstrated a p-value <0.1 in univariate analysis were entered into the multiple logistic regression model. Highly sialylated multi-branched glycans was excluded in multivariate analysis to avoid the multicollinearity with Glycan ratio <i>m/z</i> 2378/1914.</p>†<p>The data of ESR were not available in 13 patients.</p><p>Abbreviations: ALB, albumin; CAI, clinical activity index; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; PLT, platelet; WBC, white blood cell count.</p

Serum levels of glycans with different structures.

<p>The total glycan level in the serum of ulcerative colitis patients (UC, <i>red</i>) was significantly higher than that of healthy controls (HLT, <i>blue</i>), regardless of age. In particular, complex type and sialylated glycans were significantly increased in UC, although the expression of high-mannose type, hybrid type, or fucosylated glycans did not differ between UC and HLT. Lines and colored areas indicate regression curves and 95% CI, respectively.</p

Characteristics of patients with ulcerative colitis and Crohn's disease on admission and healthy volunteers.

<p>Abbreviations: ALB, albumin; CAI, clinical activity index; CD, Crohn's disease; CDAI, Crohn's disease activity index; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; HLT, healthy volunteers; IQR, interquartile range; PLT, platelet; UC, ulcerative colitis; WBC, white blood cell count.</p

Differentially expressed serum glycans with characteristics of ulcerative colitis.

<p>Box-plot of (A) the glycan ratio <i>m/z</i> 2378/1914 and (B) highly sialylated multi-branched glycans, which were significantly higher in the patients with total colitis, Clinical Activity Index >10, Mayo endoscopic score 3, or a steroid-refractory status, compared with left-sided colitis, Clinical Activity Index ≤10, Mayo endoscopic score 1–2, or no steroid-refractory status, respectively.</p