Cell-surface tethered promiscuous biotinylators enable small-scale surface proteomics of human exosomes

Characterization of cell surface proteome differences between cancer and healthy cells is a valuable approach for the identification of novel diagnostic and therapeutic targets. However, selective sampling of surface proteins for proteomics requires large samples (>10e7 cells) and long labeling times. These limitations preclude analysis of material-limited biological samples or the capture of rapid surface proteomic changes. Here, we present two labeling approaches to tether exogenous peroxidases (APEX2 and HRP) directly to cells, enabling rapid, small-scale cell surface biotinylation without the need to engineer cells. We used a novel lipidated DNA-tethered APEX2 (DNA-APEX2), which upon addition to cells promoted cell agnostic membrane-proximal labeling. Alternatively, we employed horseradish peroxidase (HRP) fused to the glycan binding domain of wheat germ agglutinin (WGA-HRP). This approach yielded a rapid and commercially inexpensive means to directly label cells containing common N-Acetylglucosamine (GlcNAc) and sialic acid glycans on their surface. The facile WGA-HRP method permitted high surface coverage of cellular samples and enabled the first comparative surface proteome characterization of cells and cell-derived exosomes, leading to the robust quantification of 1,020 cell and exosome surface proteins. We identified a newly-recognized subset of exosome-enriched markers, as well as proteins that are uniquely upregulated on Myc oncogene-transformed prostate cancer exosomes. These two cell-tethered enzyme surface biotinylation approaches are highly advantageous for rapidly and directly labeling surface proteins across a range of material-limited sample types.

Effect of thimerosal on thyroid hormones metabolism in rats

Mercury seems to exert an inhibitory effect on deiodinases, but there are few studies using Thimerosal (TM) as the mercury source. We aimed to elucidate the effect of TM on thyroid hormones peripheral metabolism. Adult Wistar female rats received 0.25 µg or 250 µg TM/100 g BW, IM, twice a week, for a month. We evaluated serum total T3 and T4, D1 activity using 125I-rT3 as tracer, and D2 activity using 125I-T4. NADPH oxidase activity was measured by Amplex-red/HRP method and mRNA levels by real time PCR. Serum T4 was increased and T3 decreased by the greatest dose of TM. Even though D1 activity in pituitary and kidney was reduced by the highest dose of TM, hepatic D1 activity and D1 mRNA levels remained unchanged. D2 activity was also significantly decreased by the highest dose of TM in all CNS samples tested, except cerebellum, but D2 mRNA was unaltered. mRNA levels of the tested NADPH oxidases were not affected by TM and NADPH oxidase activity was either unaltered or decreased. Our results indicate that TM might directly interact with deiodinases, inhibiting their activity probably by binding to their selenium catalytic site, without changes in enzyme expression.

Modified Halfspace-Relaxation Projection Methods for Solving the Split Feasibility Problem

This paper presents modified halfspace-relaxation projection (HRP) methods for solving the split feasibility problem (SFP). Incorporating with the techniques of identifying the optimal step length with positive lower bounds, the new methods improve the efficiencies of the HRP method (Qu and Xiu (2008)). Some numerical results are reported to verify the computational preference.

SCIATIC NERVE

Objective: To determine the number, size, somatotopy and segmental distribution of HRP labeledmotor and sensory neurons forming sciatic nerve in albino rat by using HRP technique. To fined out the distribution ofneurons in sciatic nerve in albino rat in spinal cord from L3S1. The average number, size and segmental distribution ofmotor and sensory neurons were localized by HRP method of tracing neuronal connections. The motor neurons formingSCN ranged 10-60 microns and extended between the caudal part of L3 and rostral part of SI spinal segment. Theyoccupied PPL, PL, C and aL subgroups. The peak frequency distribution of motor neurons was observed in L4-L6spinal segment in SCN. The labeled sensory neurons whose peripheral process run in SCN were localized in L3-S1ipsilateral Dorsal Root Ganglia (DRG). No somatotopic organization of the cells was found in the DRG. The cells weredistributed throughout the ganglia without forming groups. The somal diameters of sensory neurons forming SCNmeasured between 14-58 microns.

Mediolateral Differential Projection to the Nodulus from the Inferior Olive in the Albino Rat: A Retrograde WGA-HRP Study

The mediolateral topographic organization of olivocerebellar projections to the nodulus in rats was investigated by a retrograde WGA-HRP method. The nodulus received olivary afferents from the contralateral dorsal cap, nucleus β, ventrolateral outgrowth, and dorsomedial cell column. The mediolateral extent of terminal areas of the olivocerebellar fibers arising from these subnuclei differed. The dorsal cap projected to the entire mediolateral area of the contralateral nodulus; the rostral and caudal parts of the dorsal cap projected to the lateral and medial parts of the nodulus, respectively. The nucleus β projected to the medial and intermediate parts of the nodulus. Differential projections from the nucleus β were observed in a rostrocaudal direction as those from the dorsal cap; the rostral and medial parts of the nucleus projected to the intermediate and medial parts of the nodulus, respectively. The ventrolateral outgrowth projected only to the intermediate part of the nodulus. The dorsomedial cell column projected to the lateral half of the nodulus, including its lateral tip. These and previous findings are compared with respect to this mediolateral topography.