Factor IX San Dimas. Substitution of glutamine for Arg-4 in the propeptide leads to incomplete gamma-carboxylation and altered phospholipid binding properties.

DNA sequence analysis of the Factor IX gene from a hemophilia B patient (98% Factor IX antigen; less than 0.01 unit/ml clotting activity) has identified a point mutation in exon II. A guanine to adenine transition causes the substitution of a glutamine codon for an arginine codon at -4 in the propeptide of Factor IX. This variant, termed Factor IX San Dimas, circulates in the plasma as proFactor IX with a mutant 18-amino acid propeptide still attached. Like Factor IX Cambridge (Arg-1----Ser), Factor IX San Dimas is unable to express metal-induced epitopes recognized by conformation-specific polyclonal antibodies. Amino acid analysis of the alkaline hydrolysate indicates that purified Factor IX San Dimas contains a reduced number of gamma-carboxyglutamyl residues compared to Factor IX. However, this protein undergoes metal-induced quenching of the intrinsic fluorescence. In addition, Factor IX San Dimas is unable to interact with phospholipid vesicles. The absence of coagulant activity in Factor IX San Dimas can be attributed to impaired calcium-induced conformational changes and loss in the ability to bind phospholipid vesicles in the presence of calcium ions

Advanced Fluorescence Microscopy Techniques-FRAP, FLIP, FLAP, FRET and FLIM

Fluorescence microscopy provides an efficient and unique approach to study fixed and living cells because of its versatility, specificity, and high sensitivity. Fluorescence microscopes can both detect the fluorescence emitted from labeled molecules in biological samples as images or photometric data from which intensities and emission spectra can be deduced. By exploiting the characteristics of fluorescence, various techniques have been developed that enable the visualization and analysis of complex dynamic events in cells, organelles, and sub-organelle components within the biological specimen. The techniques described here are fluorescence recovery after photobleaching (FRAP), the related fluorescence loss in photobleaching (FLIP), fluorescence localization after photobleaching (FLAP), Forster or fluorescence resonance energy transfer (FRET) and the different ways how to measure FRET, such as acceptor bleaching, sensitized emission, polarization anisotropy, and fluorescence lifetime imaging microscopy (FLIM). First, a brief introduction into the mechanisms underlying fluorescence as a physical phenomenon and fluorescence, confocal, and multiphoton microscopy is given. Subsequently, these advanced microscopy techniques are introduced in more detail, with a description of how these techniques are performed, what needs to be considered, and what practical advantages they can bring to cell biological research

AFP, PIVKAII, GP3, SCCA-1 and follisatin as surveillance biomarkers for hepatocellular cancer in non-alcoholic and alcoholic fatty liver disease

<p>Abstract</p> <p>Background</p> <p>The incidence and mortality of hepatocellular cancer (HCC) complicating alcoholic and non-alcoholic fatty liver diseases (ALD and NAFLD) is rising in western societies. Despite knowing the at risk populations for HCC development, the lack of sensitive and specific means of surveillance hampers disease detection at curable stages. The most widely used serum HCC marker is alpha-fetoprotein (AFP), while PIVKA-II, glypican-3 (GP3) and Squamous Cell Carcinoma Antigen -1 (SCCA-1) have been proposed as new biomarkers. Assessment of these HCC biomarkers has largely been performed in patients with viral hepatitis. We conducted a cross sectional study assessing the value of these serum proteins, as well a novel candidate biomarker -follistatin – in patients with HCC arising on a background of ALD or NAFLD.</p> <p>Methods</p> <p>Pre-treatment serum samples from 50 patients with HCC arising on a background of ALD (n = 31) or NAFLD (n = 19) were assessed by specific ELISA assay for PIVKAII, Glypican-3, SCCA-1 and Follistatin. Results were compared and contrasted with a control patient group with biopsy proven steatohepatitis-related cirrhosis (n = 41). The diagnostic accuracy of each of the candidate biomarkers was evaluated using receiver operating characteristic (ROC) curve analysis, reporting the area under the curve (AUC) and its 95% confidence interval (CI). Performance was compared to that of the established biomarker, AFP.</p> <p>Results</p> <p>Serum levels of all proteins were assessed by specific ELISA assays. GP3, SCCA-1 and follistatin had no HCC surveillance benefit in these patients. AFP and PIVKAII were superior to the other markers, particularly in combination.</p> <p>Conclusion</p> <p>We conclude that while novel means of surveillance are urgently required, the combination of AFP and PIVKAII for HCC is an improvement on AFP alone in ALD/NAFLD patients. Furthermore, our data in this homogenous subset of patients- particularly that confirming no role for SCCA-1 – suggests that the choice of optimal biomarkers for HCC surveillance may be determined by the aetiology of underlying chronic liver disease.</p

Light-adapted spectral sensitivity of the goldfish: A reflex measure

Light-adapted spectral sensitivity of the goldfish was measured using the dorsal light reaction--the reflexive tendency of fish to tilt their dorsal surface toward bright light. Subjects were placed between two 150[deg] extended monochromatic fields, one of which was always 532 nm. The angle of tilt toward or away from the 532 nm light was measured, and the relative intensities of the fields were adjusted until the fish did not tilt. Blind fish did not tilt at all, and fish without utricular otoliths tilted more than normal. The spectral sensitivity obtained using this method peaked near 600 nm and was shallow and broad. The results are similar to those of electrophysiological studies, but different from other behavioral studies.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/22775/1/0000330.pd

Evaluation of patients with a recent clinical fracture and osteoporosis, a multidisciplinary approach

The aetiology of osteoporotic fractures is multifactorial, but little is known about the way to evaluate patients with a recent clinical fracture for the presence of secondary osteoporosis

Spatial Dimensions of Dengue Virus Transmission across Interepidemic and Epidemic Periods in Iquitos, Peru (1999–2003)

To target prevention and control strategies for dengue fever, it is essential to understand how the virus travels through the city. We report spatial analyses of dengue infections from a study monitoring school children and adult family members for dengue infection at six-month intervals from 1999–2003, in the Amazonian city of Iquitos, Peru. At the beginning of the study, only DENV serotypes 1 and 2 were circulating. Clusters of infections of these two viruses were concentrated in the northern region of the city, where mosquito indices and previous DENV infection were both high. In 2002, DENV-3 invaded the city, replacing DENV-1 and -2 as the dominant strain. During the invasion process, the virus spread rapidly across the city, at low levels. After this initial phase, clusters of infection appeared first in the northern region of the city, where clusters of DENV-1 and DENV-2 had occurred in prior years. Most of the clusters we identified had radii >100 meters, indicating that targeted or reactive treatment of these high-risk areas might be an effective proactive intervention strategy. Our results also help explain why vector control within 100 m of a dengue case is often not successful for large-scale disease prevention

Distinct Type of Transmission Barrier Revealed by Study of Multiple Prion Determinants of Rnq1

Prions are self-propagating protein conformations. Transmission of the prion state between non-identical proteins, e.g. between homologous proteins from different species, is frequently inefficient. Transmission barriers are attributed to sequence differences in prion proteins, but their underlying mechanisms are not clear. Here we use a yeast Rnq1/[PIN+]-based experimental system to explore the nature of transmission barriers. [PIN+], the prion form of Rnq1, is common in wild and laboratory yeast strains, where it facilitates the appearance of other prions. Rnq1's prion domain carries four discrete QN-rich regions. We start by showing that Rnq1 encompasses multiple prion determinants that can independently drive amyloid formation in vitro and transmit the [PIN+] prion state in vivo. Subsequent analysis of [PIN+] transmission between Rnq1 fragments with different sets of prion determinants established that (i) one common QN-rich region is required and usually sufficient for the transmission; (ii) despite identical sequences of the common QNs, such transmissions are impeded by barriers of different strength. Existence of transmission barriers in the absence of amino acid mismatches in transmitting regions indicates that in complex prion domains multiple prion determinants act cooperatively to attain the final prion conformation, and reveals transmission barriers determined by this cooperative fold

Circulating microparticles: square the circle

Background: The present review summarizes current knowledge about microparticles (MPs) and provides a systematic overview of last 20 years of research on circulating MPs, with particular focus on their clinical relevance. Results: MPs are a heterogeneous population of cell-derived vesicles, with sizes ranging between 50 and 1000 nm. MPs are capable of transferring peptides, proteins, lipid components, microRNA, mRNA, and DNA from one cell to another without direct cell-to-cell contact. Growing evidence suggests that MPs present in peripheral blood and body fluids contribute to the development and progression of cancer, and are of pathophysiological relevance for autoimmune, inflammatory, infectious, cardiovascular, hematological, and other diseases. MPs have large diagnostic potential as biomarkers; however, due to current technological limitations in purification of MPs and an absence of standardized methods of MP detection, challenges remain in validating the potential of MPs as a non-invasive and early diagnostic platform. Conclusions: Improvements in the effective deciphering of MP molecular signatures will be critical not only for diagnostics, but also for the evaluation of treatment regimens and predicting disease outcomes