Limitations of methods for measuring the concentration of human genomic DNA and oligonucleotide samples.

We compared different methods (absorbance, fluorescent dye-binding, and digital PCR) for measuring the concentrations of human genomic DNA from cultured cells and absorbance measurements of a synthetic DNA oligonucleotide. NIST Standard Reference Material (SRM) 2082, a pathlength absorbance standard, was used to benchmark the absorbance measurements done with microvolume spectrophotometers and a microvolume plate reader. Control absorbance values were measured on a high accuracy spectrophotometer and a NIST calibrated pathlength cuvette.

NIST Spectroscopic Measurement Standards.

Ultraviolet (UV) absorbance measurements provide a rapid and reliable method to determine protein concentrations. the National Institute of standards and technology (NIST) has developed as a pathlength standard for UV absorbance measurements for use with the new generation of microvolume spectrophotometers and short-pathlength cuvettes. short pathlengths are used with high-concentration targets to ensure that absorbance values are within the optimal range.

Steps to achieve quantitative measurements of microRNA using two step droplet digital PCR.

Droplet digital PCR (ddPCR) is being advocated as a reference method to measure rare genomic targets. It has consistently been proven to be more sensitive and direct at discerning copy numbers of DNA than other quantitative methods. However, one of the largest obstacles to measuring microRNA (miRNA) using ddPCR is that reverse transcription efficiency depends upon the target, meaning small RNA nucleotide composition directly effects primer specificity in a manner that prevents traditional quantitation optimization strategies.

Secreted Thrombospondin-1 Regulates Macrophage Interleukin-1β Production and Activation through CD47.

Thrombospondin-1 regulates inflammation by engaging several cell surface receptors and by modulating activities of other secreted factors. We have uncovered a novel role of thrombospondin-1 in modulating production and activation of the proinflammatory cytokine IL-1β by human and murine macrophages. Physiological concentrations of thrombospondin-1 limit the induction by lipopolysaccharide of IL-1β mRNA and total protein production by human macrophages.

CD47 Promotes Protective Innate and Adaptive Immunity in a Mouse Model of Disseminated Candidiasis.

CD47 is a widely expressed receptor that regulates immunity by engaging its counter-receptor SIRPα on phagocytes and its secreted ligand thrombospondin-1. Mice lacking CD47 can exhibit enhanced or impaired host responses to bacterial pathogens, but its role in fungal immunity has not been examined. cd47-/- mice on a C57BL/6 background showed significantly increased morbidity and mortality following Candida albicans infection when compared with wild-type mice.

Thrombospondin-1 signaling through CD47 inhibits self-renewal by regulating c-Myc and other stem cell transcription factors.

Signaling through the thrombospondin-1 receptor CD47 broadly limits cell and tissue survival of stress, but the molecular mechanisms are incompletely understood. We now show that loss of CD47 permits sustained proliferation of primary murine endothelial cells, increases asymmetric division, and enables these cells to spontaneously reprogram to form multipotent embryoid body-like clusters. c-Myc, Klf4, Oct4, and Sox2 expression is elevated in CD47-null endothelial cells, in several tissues of CD47- and thrombospondin-1-null mice, and in a human T cell line lacking CD47.

Therapeutic opportunities for targeting the ubiquitous cell surface receptor CD47.

Introduction: CD47 is a ubiquitously expressed cell surface receptor that serves as a counter-receptor for SIRPα in recognition of self by the innate immune system. Independently, CD47 also functions as an important signaling receptor for regulating cell responses to stress.

Areas Covered: We review the expression, molecular interactions, and pathophysiological functions of CD47 in the cardiovascular and immune systems.

Hydrogen sulfide is an endogenous potentiator of T cell activation.

H(2)S is an endogenous signaling molecule that may act via protein sulfhydrylation to regulate various physiological functions. H(2)S is also a byproduct of dietary sulfate metabolism by gut bacteria. Inflammatory bowel diseases such as ulcerative colitis are associated with an increase in the colonization of the intestine by sulfate reducing bacteria along with an increase in H(2)S production.

GPR124, an orphan G protein-coupled receptor, is required for CNS-specific vascularization and establishment of the blood-brain barrier.

Every organ in the body requires blood vessels for efficient delivery of oxygen and nutrients, but independent vascular beds are highly specialized to meet the individual needs of specific organs. The vasculature of the brain is tightly sealed, with blood-brain barrier (BBB) properties developing coincident with neural vascularization. G protein-coupled receptor 124 (GPR124) (tumor endothelial marker 5, TEM5), an orphan member of the adhesion family of G protein-coupled receptors, was previously identified on the basis of its overexpression in tumor vasculature.

Estrogen receptor α and aromatase polymorphisms affect risk, prognosis, and therapeutic outcome in men with castration-resistant prostate cancer treated with docetaxel-based therapy.

Context: Reactive estrogen species cause genotoxicity and interfere with docetaxel-mediated tubulin polymerization resulting in shortened survival in men with castrate-resistant prostate cancer (CRPC).

Objective: We hypothesized that polymorphisms in estrogen synthesis and estrogen targets (i.e.

A membrane protein/signaling protein interaction network for Arabidopsis version AMPv2.

Interactions between membrane proteins and the soluble fraction are essential for signal transduction and for regulating nutrient transport. To gain insights into the membrane-based interactome, 3,852 open reading frames (ORFs) out of a target list of 8,383 representing membrane and signaling proteins from Arabidopsis thaliana were cloned into a Gateway-compatible vector. The mating-based split ubiquitin system was used to screen for potential protein-protein interactions (pPPIs) among 490 Arabidopsis ORFs.