Development of a bovine adenovirus type 2-based gene delivery vector /

ABSTRACT Recombinant adenoviruses are currently under intense investigation as potential gene delivery and gene expression vectors with applications in human and veterinary medicine. As part of our efforts to develop a bovine adenovirus type 2 (BAV2) based vector system, the nucleotide sequence of BAV2 was determined. Sixty-six open reading frames (ORFs) were found with the potential to encode polypeptides that were at least 50 amino acid (aa) residue long. Thirty-one of the BAV2 polypeptide sequences were found to share homology to already identified adenovirus proteins. The arrangement of the genes revealed that the BAV2 genomic organization closely resembles that of well-characterized human adenoviruses. In the course of this study, continuous propagation of BAV2 over many generations in cell culture resulted in the isolation of a BAV2 spontaneous mutant in which the E3 region was deleted. Restriction enzyme, sequencing and PCR analyses produced concordant results that precisely located the deletion and revealed that its size was exactly 1299 bp. The E3-deleted virus was plaque-purified and further propagated in cell culture. It appeared that the replication of such a virus lacking a portion of the E3 region was not affected, at least in cell culture. Attempts to rescue a recombinant BAV2 virus with the bacterial kanamycin resistance gene in the E3 region yielded a candidate as verified with extensive Southern blotting and PCR analyses. Attempts to purify the recombinant virus were not successful, suggesting that such recombinant BAV2 was helper-dependent. Ten clones containing full-length BAV2 genomes in a pWE15 cosmid vector were constructed. The infectivity of these constructs was tested by using different transfection methods. The BAV2 genomic clones did appear to be infectious only after extended incubation period. This may be due to limitations of various transfection methods tested, or biological differences between virus- and E. co//-derived BAV2 DNA.

The Elucidation of the involvement of endonuclease DNase y in reducing DNA transfection efficiency in mammalian cells

Gene therapy is predicated upon efficient gene transfer. While viral vectors are the method of choice for transformation efficiency, the immunogenicity and safety concerns remain problematic. Non-viral vectors, on the other hand, have shown high degrees of safety and are mostly non-immunogenic in nature. However, non-viral vectors usually suffer from low levels oftransformation efficiency and transgene expression. Thus, increasing transformation efficiency ofnon-viral vectors, in particular by calcium phosphate co-precipitation technique, is a way of generating a suitable vector for gene therapy and is the aim of this study. It is a long known fact that different cell lines have different transfection efficiencies regardless oftransfection methodology (Lin et a!., 1994). Using commonly available cell lines Madine-Darby Bovine Kidney (MDBK), HeLa and Human Embryonic Kidney (HEK-293), we have shown a decreasing trend ofDNase activity based on a plasmid digestion assay. From densitometry studies, as much as a 40% reduction in DNase activity was observed when comparing HEK-293 (least active) to MDBK (most active). Using various biochemical assays, it was determined that DNase y, in particular, was expressed more highly in MDBK cells than both HeLa and HEK-293. Upon cloning of the bovine DNase y gene, we utilized the sequence information to construct antisense expressing plasmids via both traditional antisense RNA (pASDGneoM) and siRNA (psiRNA-S4, psiRNA-S11 and psiRNA-S16). For the construction ofpASDGneoM, the 3' end of the DNase y was inserted in opposite orientation under a cytomegalovirus (CMV) promoter such that the expression ofRNA complementary to the DNase 2 ymRNA occurred. For siRNA plasmids, the sequence was screened to yield optimal short sequences for siRNA inhibition. The silencing ofbovine DNase y led to an increase in transfection efficiency based on traditional calcium phosphate co-precipitation technique; stable clones of siRNA-producing MDBK cell lines (psiRNA-S4 Bland psiRNA-S4 B4) both demol).strated 4-fold increases in transfection efficiency. Furthermore, serial transfection of antisense DNase y plasmid pASDGneoM and reporter pCMV-~ showed a maximum of 8-fold increase in transfection efficiency when the two separate transfections were carried out 4 hours apart (i.e. transfection ofpASDGneoM, separated by four hours, then transfection ofpCMV-~). Together, these results demonstrate the involvement ofDNase y in reducing transfection efficiency, at least by traditional calcium phosphate technique.

Extending the irrelevant sound effect beyond serial recall

The finding that serial recall perfonnance for visually presented items is impaired by concurrently presented speech or sounds is referred to as the irrelevant sound effect (lSE). The foremost explanation for the effect is based on interference with rehearsal and seriation processes. The present series of experiments demonstrates tha t neither rehearsal nor seriation processes is necessary to observe the ISE. Evidence comes from three experiments that a) allow participants to report to-be-remembered items in any order, b) eliminate rehearsal by engaging participants in a cover task and surprising them with a memory test, and c) show that surprise non-serial recognition is immune to rehearsal-based experimental manipulations that modulate the ISE in more typical serial recall tasks. Together,the results show that models that rely on rehearsal or seriation processes to account for the ISE need to be reconsidered. Results are discussed in tenns of interference with encoding of to-be-remembered material.

Respuesta funcional de Toxorhynchites theobaldi (DYAR y KNAB) sobre larvas de Aedes aegypti (LINN.) vector del dengue

Tesis (Maestría en Ciencias con Especialidad en Entomología Médica) UANL

Control del vector del dengue Aedes aegypti (l) con la participación de estudiantes de primaria como educadores de salud en la cuidad de Guadalupe Nuevo Léon

Tesis (Maestria en Ciencias con Especialidad en Entomología Médica) UANL

Comparación de la eficiencia de rociados domiciliares en ultra bajo volumen (ULV) y termonebulización para el control de poblaciones adultas del vector del dengue Aedes aegypti (L.) en una colonia de Guadalupe, Nuevo León, México

Tesis (Maestría en Ciencias con Especialidad en Entomología Médica) UANL

Bionomia de Aedes aegypti L., vector del dengue en el sur del estado de Jalisco, México

Tesis (Maestría en Ciencias con Especialidad en Entomología Médica) UANL

Bionomía del vector de la malaria Anopheles vestitipennis Dyar & Knab (Díptera: Culicidae) en la cuenca del Río Polochic, Alta Verapaz, Guatemala, C.A. [por] Jaime Abraham Juárez Sandoval

Tesis (Maestría en Ciencias con Especialidad en Entomología Médica) U.A.N.L.

Evaluación en campo del depredador Mesocyclops longisetus (Thiebaud) (Copepoda: Cyclopoidea) para el control larval del vector del dengue Aedes aegypti (L.) en Monterrey, Nuevo León [por] Norma Gorrochotegui Escalante

Tesis (Maestría en Ciencias con Especialidad en Entomología Médica) U.A.N.L.

Construcción del vector adenoviral AD-UV2

Tesis (Maestría en Ciencias con Especialidad en Biología Molecular e Ingeniería Genética) UANL, 2011.

Generación del vector adenoviral oncolítico Ad5

Tesis (Maestría en Ciencias con Especialidad en Biología Molecular e Ingeniería Genética) UANL, 2011.

Generación de un vector adenoviral para la producción de anticuerpos policlonales contra la proteína ENV del XMRV (Xenotropic murine leukemia virus-related virus).

Tesis (Maestría en Ciencias con Orientación en Biología Molecular e Ingeniería Genética) UANL, 2011.

A composite discrete traction vector.

Tesis (Maestría en Ciencias con Orientación en Ingeniería Estructural) UANL, 2013.

Estimating the Tobit Model with Serial Correlation: an Operational Approach

Several Authors Have Discussed Recently the Limited Dependent Variable Regression Model with Serial Correlation Between Residuals. the Pseudo-Maximum Likelihood Estimators Obtained by Ignoring Serial Correlation Altogether, Have Been Shown to Be Consistent. We Present Alternative Pseudo-Maximum Likelihood Estimators Which Are Obtained by Ignoring Serial Correlation Only Selectively. Monte Carlo Experiments on a Model with First Order Serial Correlation Suggest That Our Alternative Estimators Have Substantially Lower Mean-Squared Errors in Medium Size and Small Samples, Especially When the Serial Correlation Coefficient Is High. the Same Experiments Also Suggest That the True Level of the Confidence Intervals Established with Our Estimators by Assuming Asymptotic Normality, Is Somewhat Lower Than the Intended Level. Although the Paper Focuses on Models with Only First Order Serial Correlation, the Generalization of the Proposed Approach to Serial Correlation of Higher Order Is Also Discussed Briefly.

Nearly Serial Sharing Methods

A group of agents participate in a cooperative enterprise producing a single good. Each participant contributes a particular type of input; output is nondecreasing in these contributions. How should it be shared? We analyze the implications of the axiom of Group Monotonicity: if a group of agents simultaneously decrease their input contributions, not all of them should receive a higher share of output. We show that in combination with other more familiar axioms, this condition pins down a very small class of methods, which we dub nearly serial.