A High-Sensitivity Method for Detection and Measurement of HMGB1 Protein Concentration by High-Affinity Binding to DNA Hemicatenanes

BACKGROUND: Protein HMGB1, an abundant nuclear non-histone protein that interacts with DNA and has an architectural function in chromatin, was strikingly shown some years ago to also possess an extracellular function as an alarmin and a mediator of inflammation. This extracellular function has since been actively studied, both from a fundamental point of view and in relation to the involvement of HMGB1 in inflammatory diseases. A prerequisite for such studies is the ability to detect HMGB1 in blood or other biological fluids and to accurately measure its concentration. METHODOLOGY/PRINCIPAL FINDINGS: In addition to classical techniques (western blot, ELISA) that make use of specific anti-HMGB1 antibodies, we present here a new, extremely sensitive technique that is based on the fact that hemicatenated DNA loops (hcDNA) bind HMGB1 with extremely high affinity, higher than the affinity of specific antibodies, similar in that respect to DNA aptamers. DNA-protein complexes formed between HMGB1 and radiolabeled hcDNA are analyzed by electrophoresis on nondenaturing polyacrylamide gels using the band-shift assay method. In addition, using a simple and fast protocol to purify HMGB1 on the basis of its solubility in perchloric acid allowed us to increase the sensitivity by suppressing any nonspecific background. The technique can reliably detect HMGB1 at a concentration of 1 pg per microliter in complex fluids such as serum, and at much lower concentrations in less complex samples. It compares favorably with ELISA in terms of sensitivity and background, and is less prone to interference from masking proteins in serum. CONCLUSION: The new technique, which illustrates the potential of DNA nanoobjects and aptamers to form high-affinity complexes with selected proteins, should provide a valuable tool to further investigate the extracellular functions of HMGB1 and its involvement in inflammatory pathologies

Overview of homocysteine and folate metabolism. With special references to cardiovascular disease and neural tube defects

This overview addresses homocysteine and folate metabolism. Its functions and complexity are described, leading to explanations why disturbed homocysteine and folate metabolism is implicated in many different diseases, including congenital birth defects like congenital heart disease, cleft lip and palate, late pregnancy complications, different kinds of neurodegenerative and psychiatric diseases, osteoporosis and cancer. In addition, the inborn errors leading to hyperhomocysteinemia and homocystinuria are described. These extreme human hyperhomocysteinemia models provide knowledge about which part of the homocysteine and folate pathways are linked to which disease. For example, the very high risk for arterial and venous occlusive disease in patients with severe hyperhomocysteinemia irrespective of the location of the defect in remethylation or transsulphuration indicates that homocysteine itself or one of its “direct” derivatives is considered toxic for the cardiovascular system. Finally, common diseases associated with elevated homocysteine are discussed with the focus on cardiovascular disease and neural tube defects

Novel biomarker combination improves the diagnosis of serious bacterial infections in Malawian children

<p>Abstract</p> <p>Background</p> <p>High throughput technologies offer insight into disease processes and heightens opportunities for improved diagnostics. Using transcriptomic analyses, we aimed to discover and to evaluate the clinical validity of a combination of reliable and functionally important biomarkers of serious bacterial infection (SBI).</p> <p>Methods</p> <p>We identified three previously reported biomarkers of infection (neutrophil gelatinase-associated lipocalin (NGAL), granulysin and resistin) and measured gene expression using quantitative real-time PCR. Protein products related to the three transcripts were measured by immunoassays.</p> <p>Results</p> <p>Relative gene expression values of NGAL and resistin were significantly increased, and expression of granulysin significantly decreased in cases compared to controls. Plasma concentrations of NGAL and resistin were significantly increased in children with confirmed SBI compared to children with no detectable bacterial infection (NBI), and to controls (287 versus 128 versus 62 ng/ml and 195 versus 90 versus 18 ng/ml, respectively, p < 0.05). Plasma protein concentrations of NGAL and resistin were significantly increased in non-survivors compared to survivors (306 versus 211 and 214 versus 150 ng/ml, p = 0.02). The respective areas under the curve (AUC) for NGAL, resistin and procalcitonin in predicting SBI were 0.79, 0.80 and 0.86, whilst a combination of NGAL, resistin and procalcitonin achieved an AUC of 0.90.</p> <p>Conclusions</p> <p>We have demonstrated a unique combination of diagnostic biomarkers of SBI using transcriptomics, and demonstrated translational concordance with the corresponding protein. The addition of NGAL and resistin protein measurement to procalcitonin significantly improved the diagnosis of SBI.</p