Real-time interferometric refractive index change measurement for the direct detection of enzymatic reactions and the determination of enzyme kinetics

Back scatter interferometry (BSI) is a sensitive method for detecting changes in the bulk refractive index of a solution in a microfluidic system. Here we demonstrate that BSI can be used to directly detect enzymatic reactions and, for the first time, derive kinetic parameters. While many methods in biomedical assays rely on detectable biproducts to produce a signal, direct detection is possible if the substrate or the product exert distinct differences in their specific refractive index so that the total refractive index changes during the enzymatic reaction. In this study, both the conversion of glucose to glucose-6-phosphate, catalyzed by hexokinase, and the conversion of adenosine-triphosphate to adenosine di-phosphate and mono-phosphate, catalyzed by apyrase, were monitored by BSI. When adding hexokinase to glucose solutions containing adenosine-triphosphate, the conversion can be directly followed by BSI, which shows the increasing refractive index and a final plateau corresponding to the particular concentration. From the initial reaction velocities, KM was found to be 0.33 mM using Michaelis⁻Menten kinetics. The experiments with apyrase indicate that the refractive index also depends on the presence of various ions that must be taken into account when using this technique. This study clearly demonstrates that measuring changes in the refractive index can be used for the direct determination of substrate concentrations and enzyme kinetics

Liver cirrhosis, other liver diseases, and risk of hospitalisation for intracerebral haemorrhage: A Danish population-based case-control study

<p>Abstract</p> <p>Background</p> <p>Liver diseases are suspected risk factors for intracerebral haemorrhage (ICH). We conducted a population-based case-control study to examine risk of ICH among hospitalised patients with liver cirrhosis and other liver diseases.</p> <p>Methods</p> <p>We used data from the hospital discharge registries (1991–2003) and the Civil Registration System in Denmark, to identify 3,522 cases of first-time hospitalisation for ICH and 35,173 sex- and age-matched population controls. Among cases and controls we identified patients with a discharge diagnosis of liver cirrhosis or other liver diseases before the date of ICH. We computed odds ratios for ICH by conditional logistic regressions, adjusting for a number of confounding factors.</p> <p>Results</p> <p>There was an increased risk of ICH for patients with alcoholic liver cirrhosis (adjusted OR = 4.8, 95% CI: 2.7–8.3), non-alcoholic liver cirrhosis (adjusted OR = 7.7, 95% CI: 2.0–28.9) and non-cirrhotic alcoholic liver disease (adjusted OR = 5.4, 95%CI:3.1–9.5) but not for patients with non-cirrhotic non-alcoholic liver diseases (adjusted OR = 0.9, 95%CI:0.5–1.6). The highest risk was found among women with liver cirrhosis (OR = 8.9, 95%CI:2.9–26.7) and for patients younger than 70 years (OR = 6.1, 95%CI:3.4–10.9). There were no sex- or age-related differences in the association between other liver diseases (alcoholic or non-alcoholic) and hospitalisation with ICH.</p> <p>Conclusion</p> <p>Patients with liver cirrhosis and non-cirrhotic alcoholic liver disease have a clearly increased risk for ICH.</p

Hepatitis C prevalence in Denmark -an estimate based on multiple national registers

Background: A national survey for chronic hepatitis C has not been performed in Denmark and the prevalence is unknown. Our aim was to estimate the prevalence of chronic hepatitis C from public registers and the proportion of these patients who received specialized healthcare. Methods: Patients with a diagnosis of chronic hepatitis C were identified from four national registers: a laboratory register, the Hospital Discharge Register, a clinical database of chronic viral hepatitis and the Register of Communicable Diseases. The total population diagnosed with hepatitis C was estimated by capture-recapture analysis. The population with undiagnosed hepatitis C was derived from the national register of drug users by comparing diagnosed and tested persons. Results: A total of 6,935 patients diagnosed with chronic hepatitis C were identified in the four registers and the estimated population diagnosed with the disease was 9,166 persons (95% C.I. interval 8,973 – 9,877), corresponding to 0.21% (95% CI 0.21%-0.23%) of the Danish population over 15years of age. The prevalence was highest among persons 40–49years old (0.39%) and males (0.28%). It was estimated that 40% of the diagnosed patients lived in the capital region, and 33.5% had attended specialised healthcare. It was estimated that 46% of hepatitis C patients had not been diagnosed and the total population with chronic hepatitis C in Denmark was 16,888 (95% C.I. 16,474-18,287), corresponding to 0.38% (95% CI 0.37-0.42) of the population over 15years of age. Conclusions: The estimated prevalence of chronic hepatitis C in Denmark was 0.38%. Less than half of the patients with chronic hepatitis C in Denmark have been identified and among these patients, one in three has attended specialised care

Identification of a Shared Genetic Susceptibility Locus for Coronary Heart Disease and Periodontitis

Recent studies indicate a mutual epidemiological relationship between coronary heart disease (CHD) and periodontitis. Both diseases are associated with similar risk factors and are characterized by a chronic inflammatory process. In a candidate-gene association study, we identify an association of a genetic susceptibility locus shared by both diseases. We confirm the known association of two neighboring linkage disequilibrium regions on human chromosome 9p21.3 with CHD and show the additional strong association of these loci with the risk of aggressive periodontitis. For the lead SNP of the main associated linkage disequilibrium region, rs1333048, the odds ratio of the autosomal-recessive mode of inheritance is 1.99 (95% confidence interval 1.33–2.94; P = 6.9×10−4) for generalized aggressive periodontitis, and 1.72 (1.06–2.76; P = 2.6×10−2) for localized aggressive periodontitis. The two associated linkage disequilibrium regions map to the sequence of the large antisense noncoding RNA ANRIL, which partly overlaps regulatory and coding sequences of CDKN2A/CDKN2B. A closely located diabetes-associated variant was independent of the CHD and periodontitis risk haplotypes. Our study demonstrates that CHD and periodontitis are genetically related by at least one susceptibility locus, which is possibly involved in ANRIL activity and independent of diabetes associated risk variants within this region. Elucidation of the interplay of ANRIL transcript variants and their involvement in increased susceptibility to the interactive diseases CHD and periodontitis promises new insight into the underlying shared pathogenic mechanisms of these complex common diseases

Interactions of Adiponectin and Lipopolysaccharide from Porphyromonas gingivalis on Human Oral Epithelial Cells

BACKGROUND: Periodontitis is an inflammatory disease caused by pathogenic microorganisms, such as Porphyromonas gingivalis, and characterized by the destruction of the periodontium. Obese individuals have an increased risk for periodontitis and show decreased serum levels of adiponectin. This in-vitro study was established to examine whether adiponectin modulates critical effects of lipopolysaccharide (LPS) from P. gingivalis on oral epithelial cells (OECs). METHODOLOGY/PRINCIPAL FINDINGS: The presence of adiponectin and its receptors in human gingival tissue samples and OECs was analyzed by immunohistochemistry and PCR. Furthermore, OECs were treated with LPS and/or adiponectin for up to 72 h, and the gene expression and protein synthesis of pro- and anti-inflammatory mediators, matrix metalloproteinases (MMPs) and growth factors were analyzed by real-time PCR and ELISA. Additionally, cell proliferation, differentiation and in-vitro wound healing were studied. The nuclear translocation of NFκB was investigated by immunofluorescence. Gingival tissue sections showed a strong synthesis of adiponectin and its receptors in the epithelial layer. In cell cultures, LPS induced a significant up-regulation of interleukin (IL) 1β, IL6, IL8, MMP1 and MMP3. Adiponectin abrogated significantly the stimulatory effects of LPS on these molecules. Similarly, adiponectin inhibited significantly the LPS-induced decrease in cell viability and increase in cell proliferation and differentiation. Adiponectin led to a time-dependent induction of the anti-inflammatory mediators IL10 and heme oxygenase 1, and blocked the LPS-stimulated NFκB nuclear translocation. CONCLUSIONS/SIGNIFICANCE: Adiponectin may counteract critical actions of P. gingivalis on oral epithelial cells. Low levels of adiponectin, as observed in obese individuals, may increase the risk for periodontal inflammation and destruction