• Assessment of novel rapid assays to accurately diagnose Hepatitis C Virus infection

      Adimorah, Samuel; Vucenik, Ivana; Constantine, Niel T. (2014)
      The World Health Organization (WHO) estimates that more than 185 million people globally are infected with HCV of whom 350,000 people die annually. The diagnosis of HCV is based primarily on the use of assays that require instrumentation and cannot be performed in point of care venues. Rapid and novel HCV tests are being introduced that offer advantages, including the use of oral fluids, and some that can detect HIV and HCV simultaneously. The objectives of this study are: (1) to evaluate the diagnostic sensitivity, specificity and reproducibility of four rapid tests for the accurate detection of HCV, and noting the reactivity to HIV in those tests offering multi-agent detection; (2) to determine if an HCV rapid testing algorithm can be devised using two or more of these rapid tests that provides better accuracy than one test alone, (3) to compare other characteristics of the tests such as line intensity, slow flow through, partial lines, the impact of sample integrity, and clearance of background for easy test result reading; and (4) to evaluate the tests by comparing results when read by two observers. Four rapid HCV assays, including the OraQuick®HCV Rapid Test, the Chembio DPP HIV-HCV Assay, the Chembio DPP HCV Assay, and the Multiplo rapid HBc/HIV/HCV antibody test, were assessed to determine their ability to accurately diagnose HCV in 204 archived plasma samples. The sensitivity and specificity were determined by comparing the results obtained from the candidate assays to those of an FDA approved ELISA. Reproducibility was evaluated by running one weakly reactive and one non-reactive sample on the tests for ten consecutive days and comparing the results to the reference method. The specificity of three tests was 100%, while that of one test was 98% (one false positive). The sensitivity for all four tests was 100%, and the reproducibility of all was excellent. One of the two tests that also detects HIV produced perfect results. Further studies, using a larger sample size, are needed to verify these results and evaluate an algorithm.
    • Characterization of nudix hydrolases from predatory Bdellovibrio and like-organisms

      Pizarro Dupuy, Mario Andres; Vucenik, Ivana; Pineiro, Silvia A. (2010)
      The Bdellovibrio and like organisms are motile gram negative bacteria that prey other gram negative bacteria by entering into their periplasm and feeding from the degraded host macromolecules of the prey. The Nudix hydrolases are a superfamily of phosphoanhydrases that catalyze the hydrolysis of a nucleoside diphosphate linked to different moieties, X (NUDIX). They have in common the signature sequence GX<sub>5</sub>EX<sub>7</sub>REUXEEXGU (U=I, L or V). Some examples of phenotypes associated with Nudix enzymes are dGTPases involved in preventing mutations by removing damaged nucleotides from the nucleotide pool (mutT gene) and diadenosine tetraphosphatases involved in host invasion. The Bd2220 and Bm2 ORFs from the predatory Bdellovibrio bacteriovorus HD100 and Bacteriovorax marinus SJ, respectively, have the Nudix consensus sequence. The hypothesis proposed is that these predatory microorganisms contain Nudix hydrolases in their genome that could be related to their predatory behavior. To address this hypothesis, Bd2220 and Bm2 gene, both annotated as mutT gene at the genbank, were tested through an antimutator complementation assay. Both genes were confirmed to be not orthologue to E. coli mut T gene. The Bd2220 gene was further characterized by cloning, protein purification, enzymatic assay, subcellular localization, and a genomic in-frame deletion of the Bd2220 ORF. The BD2220 enzyme, localized at the periplasmic space of the Bd cell, was found to be a dCTP pyrophosphatase with a K<sub>M</sub> of 0.199 mM. The in-frame deletion of Bd2220 gene was conducted using the suicide vector pSSK10 and counter selecting the knock out strains (∆Bd2220) with 5% sucrose. The ∆Bd2220 showed phenotypic differentiation compared with the wild type (wt) strain. The lack of BD2220 enzyme activity caused the formation of aggregations in the ∆Bd2220 strain liquid cultures during the development stage of the bdelloplast. These aggregates were dissolved with DNase I treatment. In addition, the bdelloplast progeny was reduced, plaques were smaller and viable prey cells were observed even after several days of incubation. The proposed model is that formation of aggregates is due by the leaking of unprocessed DNA from the bdelloplast, which acted as extracellular DNA, inducing aggregation. All these findings suggest that the BD2220 enzyme is involved in a mechanism to process DNA during the bdelloplast development of the predatory life cycle.
    • DNA Methyltransferase 3B Expression is Regulated by microRNA-222 in Intestinal Epithelial Cells

      Wu, Jing; Vucenik, Ivana; Wang, Jiangying (2015)
      DNA methyltransferases (DNMTs) regulate distinct cellular functions by regulating gene transcription, genome stability, chromatin compaction and genome defense, but the exact mechanisms that control DNMTs levels remain largely unknown. Posttranscriptional processes are major mechanisms by which mammalian cells control gene expression, particularly in altering mRNA turnover and translation. microRNAs (miRNAs) are able to directly bind to the cis-elements located at the 3'-untranslated regions (3′-UTRs) of target mRNAs leading to changes in mRNA stability and translation, through which regulate gene expression. In the present study we reported that DNMT3B mRNA is a novel target of microRNA-222 (miR-222) and that miR-222 represses DNMT3B expression post-transcriptionally in human intestinal epithelial cells (IECs). Overexpression of miR-222 resulted in no significantly change in the levels of DNMT3B mRNA, but reduced new synthesis of DNMT3B dramatically. Overexpression of miR-222 also repressed the growth of intestinal epithelial cells, which arrested in G1 phase. These findings indicate that miR-222 represses translation of DNMT3B mRNA, in turn affecting intestinal epithelial homoeostasis by affecting intestinal epithelial cell proliferation.
    • Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Protects the Ovary of Mice Against Mitoxantrone Cytotoxicity

      Nnabue, Thaddeus; Merchenthaler, Istvan; Vucenik, Ivana (2016)
      PACAP is a pleiotropic polypeptide with cytotrophic, anti-inflammatory, and anti-apoptotic activities. Mitoxantrone (MTX) is a chemotherapeutic drug used for treatment of multiple sclerosis. My hypothesis is that PACAP will protect the granulosa cells of the ovarian follicle from cytotoxic cell death caused by MTX treatment. A total number of 27 mice were assigned to 3 groups; MTX, MTX + PACAP, and control. TUNEL staining, caspase-3 assay, and H&E staining were used to visualize the granulosa cells in ovary sections. H&E staining showed that MTX treatment reduced the number of developing follicles. Positive TUNEL in granulosa cells of ovary treated with MTX confirmed cell damage by MTX. Follicles in the MTX + PACAP group were TUNEL negative. The caspase-3 staining was negative in all groups. These observations suggest that MTX causes granulosa cell death from DNA damage but not due to apoptosis, and PACAP protects the granulosa cells from DNA damage.
    • Structure, Stability, and Interaction of Fibrin αC-Domain Polymers

      Mahid, Ariza; Vucenik, Ivana (2011)
      Abstract Title of the thesis: Structure, Stability, and Interaction of Fibrin αC-Domain Polymers Ariza Mahid, Master of Science, 2011 Thesis directed by: Leonid Medved Ph.D., Sc.D., Professor, Department of Biochemistry and Molecular Biology, and Center for Vascular and Inflammatory Diseases Fibrinogen is a multidomain plasma protein that after conversion into polymeric fibrin protects damaged vasculature from blood loss and subsequently participates in wound healing through the interaction of its individual domains with various plasma proteins and cellular receptors. Fibrin(ogen)αC-domains formed by the C-terminal portions of its Aαchains (amino acid residues Aα392-610) play an important role in fibrin assembly, fibrinolysis, and angiogenesis. These domains are inert in fibrinogen and highly reactive in fibrin suggesting that their structure in the latter, in which they form αC polymers, differs from that in fibrinogen. While the structure of the isolated αC-domain has been established, the structure of the αC-domains in fibrin αC polymers remains to be determined. The major goals of the present study were to clarify the structure of fibrin αC-domain polymers and to test our hypothesis that polymerization of the αC-domains results in the exposure of their binding sites. To accomplish these goals, we prepared a recombinant αC region (Aα221-610) including the αC-domain, demonstrated that it forms soluble oligomers in a concentration-dependent and reversible manner, and covalently cross-linked such oligomers with factor XIIIa. Cross-linked Aα221-610 oligomers were stable in solution and appeared as ordered linear arrays when analyzed by electron microscopy. Spectral studies revealed that theαC-domains in such oligomers were folded into compact structures with significant amount ofα-sheets and exhibited high thermal stability. These results indicate that cross-linked Aα221-610 oligomers are highly ordered and mimic the structure of αC polymers in fibrin. Binding studies performed by ELISA and SPR revealed that, in contrast to the monomeric αC-domain, the cross-linked oligomers exhibited prominent binding to tPA and plasminogen indicating that their tPA- and plasminogen-binding sites are exposed. In agreement, these oligomers drastically increased the rate of plasminogen activation in the chromogenic substrate assay. Thus, our study with cross-linked Aα221-610 oligomers clarified the structure of the αC-domains in fibrin αC polymers and confirmed our hypothesis that their binding sites are exposed upon polymerization. Such oligomers represent a stable, soluble model of fibrin αC polymers that can be used for future structural and functional studies of fibrin αC-domains.