Serum clusterin as a marker for diagnosing hepatocellular carcinoma

Background: Approximately 80% of patients with hepatocellular carcinoma (HCC) areuntreatable because of advanced tumor stages at presentation. Therefore, finding newer markers for screening and diagnosing HCC is of utmost importance. Clusterin (CLU) is a 449 amino acid, heterodimeric glycoprotein with a plausible role in the regeneration, migration, and anti-apoptosis of tumor cells. It has been implicated in many malignancies such as prostate and pancreatic adenocarcinomas, but its role in HCC is not well defined.Objective: We aimed to evaluate the diagnostic performance of serum CLU level in diagnosing HCC on top of hepatitis C virus-related liver cirrhosis, and comparing it to that of alpha fetoprotein (AFP).Methods: Twenty cases of apparently healthy subjects, 27 cases of hepatitis C virus-related liver cirrhosis (CHC cases), and 44 HCC cases on top of hepatitis C virus-related liver cirrhosis were included in this study. Serum CLU concentration was determined using a quantitative sandwich enzyme immunoassay technique.Results: Serum clusterin level showed a significant increase in the HCC group compared to the control group (151.96 ± 32.74 vs. 111.40 ±27.46) and to the CHC group (151.96 ± 32.74 vs. 89.12 ±31.62), while a significant decrease in serum clusterin level was found in the CHC group compared to the control group (89.12± 31.62 vs. 111.40± 27.46). Based on receiver operator characteristic curve analysis, serum AFP still surpassed serum CLU in diagnostic sensitivity (77.3% vs. 70.5%), specificity (100% vs. 90%), and positive and negative predictive values (100% vs. 86.1% and 83.3% vs. 77.6% respectively). The use of a combined parallel approach improved the diagnostic sensitivity (95.5%) and negative predictive value (95.7%) over the single use of AFP.Conclusions: Although the diagnostic performance of serum AFP outperformed that of serum CLU, their combined parallel approach improved the sensitivity which is required in screening high risk populations such as CHC patients. KEYWORDS Clusterin; Alpha fetoprotein; Tumor marker; Hepatitis C virus related liver cirrhosis; Hepatocellular carcinom

CHANGES IN SOME BIOPHYSICAL AND BIOCHEMICAL PARAMETERS IN BLOOD AND URINE OF WORKERS CHRONICALLY EXPOSED TO BENZENE

Objective: Benzene may occur naturally as a component of petroleum, or may be manufactured synthetically. It is found in the environment as a contaminant from both human activities and natural processes, posing serious bio-hazards from chronic exposure.Methods: A total of 330 individual were enrolled to study possible health hazards of benzene contamination; 265 males occupationally chronically exposed to low levels of benzene in their daily activity were compared to 65 healthy individuals of the same socio-economic standard. Benzene workers were divided between 45 workers in printing shops, 70 subjects dealing with benzene containing paints (painters), 75 subjects working in professions related to automotive work (autoworkers) and 75 car drivers.Results: benzene itself was not detected in blood or urine of all participants, but the levels of its metabolites; phenol and t,t-muconic acid, were higher in the blood and urine samples in the group of benzene-exposed workers. The results also indicate that individuals in this group are under oxidative stress. However, neither the determined liver function nor the kidney function tests showed significant deviation from controls. However, the results of the biophysical hematological parameters, including the degree of hemolysis, blood viscosity, RBCs aggregation and form factor were significantly deviated from normal.Conclusion: The deviation of the determined biochemical and biophysical parameters from normal may predispose such workers to a variety of health problems. Early correction of the oxidative stress and the hematological parameters and improvement of working conditions are necessary to prevent their progress to more serious health conditions, especially in children and young adolescents working under similar conditions.Running Title : Chronic exposure to benzene in work plac

Discovery of novel class of histone deacetylase inhibitors as potential anticancer agents

Selective inhibition of histone deacetylases (HDACs) is an important strategy in the field of anticancer drug discovery. However, lack of inhibitors that possess high selectivity toward certain HDACs isozymes is associated with adverse side effects that limits their clinical applications. We have initiated a collaborative initiatives between multi-institutions aimed at the discovery of novel and selective HDACs inhibitors. To this end, a phenotypic screening of an in-house pilot library of about 70 small molecules against various HDAC isozymes led to the discovery of five compounds that displayed varying degrees of HDAC isozyme selectivity. The anticancer activities of these molecules were validated using various biological assays including transcriptomic studies. Compounds 15, 14, and 19 possessed selective inhibitory activity against HDAC5, while 28 displayed selective inhibition of HDAC1 and HDAC2. Compound 22 was found to be a selective inhibitor for HDAC3 and HDAC9. Importantly, we discovered a none-hydroxamate based HDAC inhibitor, compound 28, representing a distinct chemical probe of HDAC inhibitors. It contains a trifluoromethyloxadiazolyl moiety (TFMO) as a non-chelating metal-binding group. The new compounds showed potent anti-proliferative activity when tested against MCF7 breast cancer cell line, as well as increased acetylation of histones and induce cells apoptosis. The new compounds apoptotic effects were validated through the upregulation of proapoptotic proteins caspases3 and 7 and downregulation of the antiapoptotic biomarkers C-MYC, BCL2, BCL3 and NFĸB genes. Furthermore, the new compounds arrested cell cycle at different phases, which was confirmed through downregulation of the CDK1, 2, 4, 6, E2F1 and RB1 proteins. Taken together, our findings provide the foundation for the development of new chemical probes as potential lead drug candidates for the treatment of cancer

GenomeRNAi: a database for cell-based RNAi phenotypes. 2009 update

The GenomeRNAi database (http://www.genomernai.org/) contains phenotypes from published cell-based RNA interference (RNAi) screens in Drosophila and Homo sapiens. The database connects observed phenotypes with annotations of targeted genes and information about the RNAi reagent used for the perturbation experiment. The availability of phenotypes from Drosophila and human screens also allows for phenotype searches across species. Besides reporting quantitative data from genome-scale screens, the new release of GenomeRNAi also enables reporting of data from microscopy experiments and curated phenotypes from published screens. In addition, the database provides an updated resource of RNAi reagents and their predicted quality that are available for the Drosophila and the human genome. The new version also facilitates the integration with other genomic data sets and contains expression profiling (RNA-Seq) data for several cell lines commonly used in RNAi experiments

Characterization of DNA polymerase X from Thermus thermophilus HB8 reveals the POLXc and PHP domains are both required for 3′–5′ exonuclease activity

The X-family DNA polymerases (PolXs) comprise a highly conserved DNA polymerase family found in all kingdoms. Mammalian PolXs are known to be involved in several DNA-processing pathways including repair, but the cellular functions of bacterial PolXs are less known. Many bacterial PolXs have a polymerase and histidinol phosphatase (PHP) domain at their C-termini in addition to a PolX core (POLXc) domain, and possess 3′–5′ exonuclease activity. Although both domains are highly conserved in bacteria, their molecular functions, especially for a PHP domain, are unknown. We found Thermus thermophilus HB8 PolX (ttPolX) has Mg2+/Mn2+-dependent DNA/RNA polymerase, Mn2+-dependent 3′–5′ exonuclease and DNA-binding activities. We identified the domains of ttPolX by limited proteolysis and characterized their biochemical activities. The POLXc domain was responsible for the polymerase and DNA-binding activities but exonuclease activity was not detected for either domain. However, the POLXc and PHP domains interacted with each other and a mixture of the two domains had Mn2+-dependent 3′–5′ exonuclease activity. Moreover, site-directed mutagenesis revealed catalytically important residues in the PHP domain for the 3′–5′ exonuclease activity. Our findings provide a molecular insight into the functional domain organization of bacterial PolXs, especially the requirement of the PHP domain for 3′–5′ exonuclease activity

Single-stranded DNA ligation and XLF-stimulated incompatible DNA end ligation by the XRCC4-DNA ligase IV complex: influence of terminal DNA sequence

The double-strand DNA break repair pathway, non-homologous DNA end joining (NHEJ), is distinctive for the flexibility of its nuclease, polymerase and ligase activities. Here we find that the joining of ends by XRCC4-ligase IV is markedly influenced by the terminal sequence, and a steric hindrance model can account for this. XLF (Cernunnos) stimulates the joining of both incompatible DNA ends and compatible DNA ends at physiologic concentrations of Mg2+, but only of incompatible DNA ends at higher concentrations of Mg2+, suggesting charge neutralization between the two DNA ends within the ligase complex. XRCC4-DNA ligase IV has the distinctive ability to ligate poly-dT single-stranded DNA and long dT overhangs in a Ku- and XLF-independent manner, but not other homopolymeric DNA. The dT preference of the ligase is interesting given the sequence bias of the NHEJ polymerase. These distinctive properties of the XRCC4-DNA ligase IV complex explain important aspects of its in vivo roles

Pilot assessment of the sensitivity of the malaria thin film

This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

Role of Toll-Like Receptor (TLR) 2 in Experimental Bacillus cereus Endophthalmitis

Bacillus cereus causes a uniquely rapid and blinding intraocular infection, endophthalmitis. B. cereus replicates in the eye, synthesizes numerous toxins, and incites explosive intraocular inflammation. The mechanisms involved in the rapid and explosive intraocular immune response have not been addressed. Because Toll-like receptors (TLRs) are integral to the initial recognition of organisms during infection, we hypothesized that the uniquely explosive immune response observed during B. cereus endophthalmitis is directly influenced by the presence of TLR2, a known Gram-positive pathogen recognition receptor. To address this hypothesis, we compared the courses of experimental B. cereus endophthalmitis in wild type C57BL/6J mice to that of age-matched homozygous TLR2-/- mice. Output parameters included analysis of bacterial growth, inflammatory cell (PMN) infiltration, cytokine/chemokine kinetics, retinal function testing, and histology, with N≥4 eyes/assay/time point/mouse strain. B. cereus grew at similar rates to108 CFU/eye by 12 h, regardless of the mouse strain. Retinal function was preserved to a greater degree in infected TLR2-/- eyes compared to that of infected wild type eyes, but infected eyes of both mouse strains lost significant function. Retinal architecture was preserved in infected TLR2-/- eyes, with limited retinal and vitreal cellular infiltration compared to that of infected wild type eyes. Ocular myeloperoxidase activities corroborated these results. In general, TNFα, IFNγ, IL6, and KC were detected in greater concentrations in infected wild type eyes than in infected TLR2-/- eyes. The absence of TLR2 resulted in decreased intraocular proinflammatory cytokine/chemokine levels and altered recruitment of inflammatory cells into the eye, resulting in less intraocular inflammation and preservation of retinal architecture, and a slightly greater degree of retinal function. These results demonstrate TLR2 is an important component of the initial ocular response to B. cereus endophthalmitis

RNAi Screening in Drosophila Cells Identifies New Modifiers of Mutant Huntingtin Aggregation

The fruitfly Drosophila melanogaster is well established as a model system in the study of human neurodegenerative diseases. Utilizing RNAi, we have carried out a high-throughput screen for modifiers of aggregate formation in Drosophila larval CNS-derived cells expressing mutant human Huntingtin exon 1 fused to EGFP with an expanded polyglutamine repeat (62Q). 7200 genes, encompassing around 50% of the Drosophila genome, were screened, resulting in the identification of 404 candidates that either suppress or enhance aggregation. These candidates were subjected to secondary screening in normal length (18Q)-expressing cells and pruned to remove dsRNAs with greater than 10 off-target effects (OTEs). De novo RNAi probes were designed and synthesized for the remaining 68 candidates. Following a tertiary round of screening, 21 high confidence candidates were analyzed in vivo for their ability to modify mutant Huntingtin-induced eye degeneration and brain aggregation. We have established useful models for the study of human HD using the fly, and through our RNAi screen, we have identified new modifiers of mutant human Huntingtin aggregation and aggregate formation in the brain. Newly identified modifiers including genes related to nuclear transport, nucleotide processes, and signaling, may be involved in polyglutamine aggregate formation and Huntington disease cascades

False negative rates in Drosophila cell-based RNAi screens: a case study

<p>Abstract</p> <p>Background</p> <p>High-throughput screening using RNAi is a powerful gene discovery method but is often complicated by false positive and false negative results. Whereas false positive results associated with RNAi reagents has been a matter of extensive study, the issue of false negatives has received less attention.</p> <p>Results</p> <p>We performed a meta-analysis of several genome-wide, cell-based <it>Drosophila </it>RNAi screens, together with a more focused RNAi screen, and conclude that the rate of false negative results is at least 8%. Further, we demonstrate how knowledge of the cell transcriptome can be used to resolve ambiguous results and how the number of false negative results can be reduced by using multiple, independently-tested RNAi reagents per gene.</p> <p>Conclusions</p> <p>RNAi reagents that target the same gene do not always yield consistent results due to false positives and weak or ineffective reagents. False positive results can be partially minimized by filtering with transcriptome data. RNAi libraries with multiple reagents per gene also reduce false positive and false negative outcomes when inconsistent results are disambiguated carefully.</p