Automated Entire Thrombus Density Measurements for Robust and Comprehensive Thrombus Characterization in Patients with Acute Ischemic Stroke

Background and Purpose

In acute ischemic stroke (AIS) management, CT-based thrombus density has been associated with treatment success. However, currently used thrombus measurements are prone to inter-observer variability and oversimplify the heterogeneous thrombus composition. Our aim was first to introduce an automated method to assess the entire thrombus density and then to compare the measured entire thrombus density with respect to current standard manual measurements.

Materials and Method

In 135 AIS patients, the density distribution of the entire thrombus was determined. Density distributions were described using medians, interquartile ranges (IQR), kurtosis, and skewedness. Differences between the median of entire thrombus measurements and commonly applied manual measurements using 3 regions of interest were determined using linear regression.

Results

Density distributions varied considerably with medians ranging from 20.0 to 62.8 HU and IQRs ranging from 9.3 to 55.8 HU. The average median of the thrombus density distributions (43.5 ± 10.2 HU) was lower than the manual assessment (49.6 ± 8.0 HU) (p<0.05). The difference between manual measurements and median density of entire thrombus decreased with increasing density (r = 0.64; p<0.05), revealing relatively higher manual measurements for low density thrombi such that manual density measurement tend overestimates the real thrombus density.

Conclusions

Automatic measurements of the full thrombus expose a wide variety of thrombi density distribution, which is not grasped with currently used manual measurement. Furthermore, discrimination of low and high density thrombi is improved with the automated method.     

The Determination of Assay for Laccase of Bacillus subtilis WPI with Two Classes of Chemical Compounds as Substrates

Ligninolytic enzyme complexes are involved in lignin degradation. Among them laccases are outstanding because they use molecular oxygen as a co-substrate instead of hydrogen peroxide as used by peroxidases. Bacterial laccase of Bacillus genus was first reported in Claus and Filip (Microbiol Res 152:209–216, 1997), since then more bacterial laccases have been found. In this research, laccase-producing bacteria were screened from pulp and paper industry wastewater, bagass and sugarcane rhizosphere. Nutrient agar medium containing 0.5 mM of guaiacol was used. It was observed that the laccase-producing strains developed brown colour from which 16 strains of Bacillus were identified. One of the isolated strains was identified as Bacillus subtilis WPI based on the results of biochemical tests and 16S rDNA sequence analysis. This strain showed laccase-like activity towards the oxidizing substrates ABTS and guaiacol. In this study guaiacol was used as the substrate of laccase activity assay. For determination of laccase activity of this isolate guaiacol was used as a substrate of assay for the first time in this study. SDS-PAGE and Native-PAGE confirmed the presence of laccase.     

In silico design and in vitro expression of novel multiepitope DNA constructs based on HIV-1 proteins and Hsp70 T-cell epitopes

Objectives

Epitope-driven vaccines carrying highly conserved and immunodominant epitopes have emerged as promising approaches to overcome human immunodeficiency virus-1 (HIV-1) infection.

Methods

Two multiepitope DNA constructs encoding T cell epitopes from HIV-1 Gag, Pol, Env, Nef and Rev proteins alone and/or linked to the immunogenic epitopes derived from heat shock protein 70 (Hsp70) as an immunostimulatory agent were designed. In silico analyses were applied including MHC-I and MHC-II binding, MHC-I immunogenicity and antigen processing, population coverage, conservancy, allergenicity, toxicity and hemotoxicity. The peptide-MHC-I/MHC-II molecular docking and cytokine production analyses were carried out for predicted epitopes. The selected highly immunogenic T-cell epitopes were then used to design two multiepitope fusion constructs. Next, prediction of the physicochemical and structural properties, B cell epitopes, and constructs-toll-like receptors (TLRs) molecular docking were performed for each construct. Finally, the eukaryotic expression plasmids harboring totally 12 cytotoxic T Lymphocyte (CTL) and 10 helper T lymphocytes (HTL) epitopes from HIV-1 proteins (i.e., pEGFP-N1-gag-pol-env-nef-rev), and linked to 2 CTL and 2 HTL epitopes from Hsp70 (i.e., pEGFP-N1-hsp70-gag-pol-env-nef-rev) were generated and transfected into HEK-293 T cells for evaluating the percentage of multiepitope peptides expression using flow cytometry and western blotting.

Results

The designed DNA constructs could be successfully expressed in mammalian cells. The expression rates of Gag-Pol-Env-Nef-Rev-GFP and Hsp70-Gag-Pol-Env-Nef-Rev-GFP were about 56–60% as the bands of?~?63 and?~?72 kDa confirmed in western blotting, respectively.

Conclusion

The combined in silico/in vitro methods indicated two multiepitope constructs can be produced and used as probable effective immunogens for HIV-1 vaccine development.

     

Towards a framework for the evolutionary genomics of Kinetoplastids: what kind of data and how much?

The current status of kinetoplastids phylogeny and evolution is discussed in view of the recent progresses on genomics. Some ideas on a potential framework for the evolutionary genomics of kinetoplastids are presented.     

The role of intravascular ultrasound imaging in vascular brachytherapy

Intracoronary brachytherapy has recently emerged as a new therapy to prevent restenosis. Initial experimental work was achieved in animal models and the results were assessed by histomorphometry. Initial clinical trials used angiography to guide dosimetry and to assess efficacy. Intravascular ultrasound (IVUS) permits tomographic examination of the vessel wall, elucidating the true morphology of the lumen and transmural components, which cannot be investigated on the lumenogram obtained by angiography. This paper reviews the use of IVUS in the clinical studies of brachytherapy conducted to date. IVUS allows clinicians to make a thorough assessment of the remodeling of the vessel and appears to have a major role to play in facilitating understanding of the underlying mechanisms of action in this emerging field. The authors propose that state-of-the-art IVUS techniques should be employed to further knowledge of the mechanisms of action of brachytherapy in atherosclerotic human coronary arteries.     

Improving model construction of profile HMMs for remote homology detection through structural alignment

Background  

Remote homology detection is a challenging problem in Bioinformatics. Arguably, profile Hidden Markov Models (pHMMs) are one of the most successful approaches in addressing this important problem. pHMM packages present a relatively small computational cost, and perform particularly well at recognizing remote homologies. This raises the question of whether structural alignments could impact the performance of pHMMs trained from proteins in the Twilight Zone, as structural alignments are often more accurate than sequence alignments at identifying motifs and functional residues. Next, we assess the impact of using structural alignments in pHMM performance.     

Salicylate poly(vinyl chloride) membrane electrode based on (2-[(E)-2-(4-nitrophenyl) hydrazono]-1-phenyl-2-(2-quinolyl)-1-ethanone) Cu(II)

A new salicylate-selective electrode based on the complex of (2-[(E)-2-(4-nitrophenyl)hydrazono]-1-phenyl-2-(2-quinolyl)-1-ethanone) Cu(II) as the membrane carrier was developed. The electrode exhibited a good Nernstian slope of -59.6+/-1.0 mV/decade and a linear range of 1.0 x 10(-6) to 1.0M for salicylate. The limit of detection was 5.0 x 10(-7) M. The electrode had a fast response time of 10 s and can be used for more than 3 months. The selective coefficients were determined by the fixed interference method and could be used in the pH range of 4.0 to 10.5. The electrode was employed as an indicator electrode for direct determination of salicylate in pharmaceutical and biological samples.     

Development and validation of a rapid HPLC method for simultaneous determination of tramadol, and its two main metabolites in human plasma

Tramadol, an analgesic agent, and its two main metabolites O-desmethyltramadol (M1) and N-desmethyltramadol (M2) were determined simultaneously in human plasma by a rapid and specific HPLC method. The sample preparation was a simple extraction with ethyl acetate. Chromatographic separation was achieved with a Chromolith Performance RP-18e 50 mm x 4.6 mm column, using a mixture of methanol:water (13:87, v/v) adjusted to pH 2.5 by phosphoric acid, in an isocratic mode at flow rate of 2 ml/min. Fluorescence detection (lambda(ex)=200 nm/lambda(em)=301 nm) was used. The calibration curves were linear (r(2)>0.997) in the concentration range of 2.5-500 ng/ml, 1.25-500 ng/ml and 5-500 ng/ml for tramadol, M1 and M2, respectively. The lower limit of quantification was 2.5 ng/ml for tramadol, 1.25 ng/ml for M1 and 5 ng/ml for M2. The within- and between-day precisions in the measurement of QC samples at four tested concentrations were in the range of 2.5-9.7%, 2.5-9.9% and 5.9-11.3% for tramadol, M1 and M2, respectively. The developed procedure was applied to assess the pharmacokinetics of tramadol and its two main metabolites following administration of 100mg single oral dose of tramadol to healthy volunteers.