Development and Utility of an Internal Threshold Control (ITC) Real-Time PCR Assay for Exogenous DNA Detection

Sensitive and specific tests for detecting exogenous DNA molecules are useful for infectious disease diagnosis, gene therapy clinical trial safety, and gene doping surveillance. Taqman real-time PCR using specific sequence probes provides an effective approach to accurately and quantitatively detect exogenous DNA. However, one of the major challenges in these analyses is to eliminate false positive signals caused by either non-targeted exogenous or endogenous DNA sequences, or false negative signals caused by impurities that inhibit PCR. Although multiplex Taqman PCR assays have been applied to address these problems by adding extra primer-probe sets targeted to endogenous DNA sequences, the differences between targets can lead to different detection efficiencies. To avoid these complications, a Taqman PCR-based approach that incorporates an internal threshold control (ITC) has been developed. In this single reaction format, the target sequence and ITC template are co-amplified by the same primers, but are detected by different probes each with a unique fluorescent dye. Sample DNA, a prescribed number of ITC template molecules set near the limit of sensitivity, a single pair of primers, target probe and ITC probe are added to one reaction. Fluorescence emission signals are obtained simultaneously to determine the cycle thresholds (Ct) for amplification of the target and ITC sequences. The comparison of the target Ct with the ITC Ct indicates if a sample is a true positive for the target (i.e. Ct less than or equal to the ITC Ct) or negative (i.e. Ct greater than the ITC Ct). The utility of this approach was demonstrated in a nonhuman primate model of rAAV vector mediated gene doping in vivo and in human genomic DNA spiked with plasmid DNA

Viral Gene Delivery Selectively Restores Feeding and Prevents Lethality of Dopamine-Deficient Mice

AbstractDopamine-deficient mice (DA−/−), lacking tyrosine hydroxylase (TH) in dopaminergic neurons, become hypoactive and aphagic and die by 4 weeks of age. They are rescued by daily treatment with L-3,4-dihydroxyphenylalanine (L-DOPA); each dose restores dopamine (DA) and feeding for less than 24 hr. Recombinant adeno-associated viruses expressing human TH or GTP cyclohydrolase 1 (GTPCH1) were injected into the striatum of DA−/− mice. Bilateral coinjection of both viruses restored feeding behavior for several months. However, locomotor activity and coordination were partially improved. A virus expressing only TH was less effective, and one expressing GTPCH1 alone was ineffective. TH immunoreactivity and DA were detected in the ventral striatum and adjacent posterior regions of rescued mice, suggesting that these regions mediate a critical DA-dependent aspect of feeding behavior

The Technological Development of Minimally Invasive Spine Surgery

Minimally invasive spine surgery has its roots in the mid-twentieth century with a few surgeons and a few techniques, but it has now developed into a large field of progressive spinal surgery. A wide range of techniques are now called \ minimally invasive,\ and case reports are submitted constantly with new \ minimally invasive\ approaches to spinal pathology. As minimally invasive spine surgery has become more mainstream over the past ten years, in this paper we discuss its history and development. © 2014 Laura A. Snyder et al

Gene delivery to in situ veins: Differential effects of adenovirus and adeno-associated viral vectors

AbstractPurpose: Gene transfer offers the potential to modify vein graft biology at the time of surgical implantation. Efficiency of gene delivery, stability of expression, and host responses are critical parameters for candidate vectors. We compared the effects of intraluminal exposure with adenovirus (AD) and adeno-associated virus (AAV) vectors on transgene expression and monocyte adhesion (MA) in treated vein segments. Methods: Adult New Zealand white rabbits (N = 51) were anesthetized, and the jugular veins were cannulated bilaterally. Veins were gently distended with either vector (2·108 to 1·1010 infective particles/mL) or vehicle (control) for 30 minutes, after which venous flow was restored. AD and AAV vectors encoding for the marker genes β-galactosidase (LacZ) and green fluorescent protein (GFP) were used. Vessels were explanted 2 to 40 days postinfection for analysis of gene expression (X-gal staining, reverse transcriptase-polymerase chain reaction), MA, and immunohistochemistry. Ex vivo adhesion assays used 51Cr-labeled THP-1 cells. Statistical significance was tested by using analysis of variance with a P value less than.05. Results: All animals survived, and all treated veins were patent at sacrifice. Intraluminal exposure to AD at a titer of 1·109 resulted in near complete transduction of the endothelium at 2 days, with no detectable expression by day 14. At an equal titer of infectious particles, transgene expression was markedly less for AAV at 2 to 7 days, but improved at 2 weeks and persisted to 40 days. MA was significantly increased 2 days after AD exposure (2.7-fold vs control, *P <.002); AAV treatment had no discernible effect on MA. Conclusion: AD-mediated gene transfer to vein segments resulted in robust, transient gene expression that disappeared after 2 weeks. In comparison, AAV-mediated gene delivery was less efficient, but resulted in delayed onset, persistent expression beyond 30 days. AD exposure induced an early increase in MA to the vein surface that was not seen with AAV treatment. Current generations of both AD and AAV vectors have significant, albeit different, limitations for vascular gene therapy. (J Vasc Surg 2000;31:1149-59.

Prion disease induced alterations in gene expression in spleen and brain prior to clinical symptoms

Prion diseases are fatal neurodegenerative disorders that affect animals and humans. There is a need to gain understanding of prion disease pathogenesis and to develop diagnostic assays to detect prion diseases prior to the onset of clinical symptoms. The goal of this study was to identify genes that show altered expression early in the disease process in the spleen and brain of prion disease-infected mice. Using Affymetrix microarrays, we identified 67 genes that showed increased expression in the brains of prion disease-infected mice prior to the onset of clinical symptoms. These genes function in many cellular processes including immunity, the endosome/lysosome system, hormone activity, and the cytoskeleton. We confirmed a subset of these gene expression alterations using other methods and determined the time course in which these changes occur. We also identified 14 genes showing altered expression prior to the onset of clinical symptoms in spleens of prion disease infected mice. Interestingly, four genes, Atp1b1, Gh, Anp32a, and Grn, were altered at the very early time of 46 days post-infection. These gene expression alterations provide insights into the molecular mechanisms underlying prion disease pathogenesis and may serve as surrogate markers for the early detection and diagnosis of prion disease

Native American Children and Their Reports of Hope: Construct Validation of the Children's Hope Scale

Child reports of hope continue to be utilized as predictors of positive adjustment; however, the utilization of the hope construct has not been assessed within the culturally diverse Native American child group. The present study investigated the applicability of the Hope theory among 96 Native American children in the Midwest. Measures included the Children’s Hope Scale and a Hope Interview. Native American children in the current sample appear to conceptualize hope as a way to reach goals as did the children in the normative sample. Results from the factor analysis demonstrate that the factor structure found in the current study was similar to the factor structure found in the standardization sample. Because of the similar Hope theory conceptualization and factor structure, interventions focused on the positive psychology construct of hope may be applicable within a Native American child population

Sanctions, Benefits, and Rights: Three Faces of Accountability

As countries throughout the world democratize and decentralize, citizen participation in public life should increase. In this paper, I suggest that democratic participation in local government is enhanced when citizens can reply affirmatively to at least three questions about their ability to hold local officials accountable for their actions: Can citizens use the vote effectively to reward and punish the general or specific performance of local public officials and/or the parties they represent? Can citizens generate response to their collective needs from local governments? Can citizens be ensured of fair and equitable treatment from public agencies at local levels? The findings of a study of 30 randomly selected municipalities in Mexico indicate that, over the course of a decade and a half, voters were able to enforce alternation in power and the circulation of elites, but not necessarily to transmit unambiguous messages to public officials or parties about performance concerns. More definitively, citizens were able to build successfully on prior political experiences to extract benefits from local governments. At the same time, the ability to demand good performance of local government as a right of citizenship lagged behind other forms of accountability

The Grizzly, November 17, 1989

Inspired Voices Speak Out Nationally • Appealing for Unborn Lives • Boorstin Speaks at U.C. • Letters: Pledging Under Siege; Grizzly Growls; Did Berman Ask You?; Doctors do it Right; Only Doug; Wipe Mud From Shoudt\u27s Face; Wrong!; GDI Promotes Disunity • Changing Dining Atmosphere • Save a Forest: Recycle! • Career Day • Running Bears Finish Strong • Grizzlies Downed by Devils • Ladies Finish Winning Season • Praise Hockey Team • Swimming Prospectives • Greek News • Stroke on A\u27Bears • Don\u27t Talk Dirty to Me • Top Ten Things Loved at U.C.https://digitalcommons.ursinus.edu/grizzlynews/1247/thumbnail.jp