BackgroundDengue viruses (DENV) are one of the most important viral diseases in the world with approximately 100 million infections and 200,000 deaths each year. The current lack of an approved tetravalent vaccine and ineffective insecticide control measures warrant a search for alternatives to effectively combat DENV. The trans-splicing variant of the Tetrahymena thermophila group I intron catalytic RNA, or ribozyme, is a powerful tool for post-transcriptional RNA modification. The nature of the ribozyme and the predictability with which it can be directed makes it a powerful tool for modifying RNA in nearly any cell type without the need for genome-altering gene therapy techniques or dependence on native cofactors.ResultsSeveral anti-DENV Group I trans-splicing introns (αDENV-GrpIs) were designed and tested for their ability to target DENV-2 NGC genomes in situ. We have successfully targeted two different uracil bases on the positive sense genomic strand within the highly conserved 5'-3' cyclization sequence (CS) region common to all serotypes of DENV with our αDENV-GrpIs. Our ribozymes have demonstrated ability to specifically trans-splice a new RNA sequence downstream of the targeted site in vitro and in transfected insect cells as analyzed by firefly luciferase and RT-PCR assays. The effectiveness of these αDENV-GrpIs to target infecting DENV genomes is also validated in transfected or transformed Aedes mosquito cell lines upon infection with unattenuated DENV-2 NGC.ConclusionsAnalysis shows that our αDENV-GrpIs have the ability to effectively trans-splice the DENV genome in situ. Notably, these results show that the αDENV-GrpI 9v1, designed to be active against all forms of Dengue virus, effectively targeted the DENV-2 NGC genome in a sequence specific manner. These novel αDENV-GrpI introns provide a striking alternative to other RNA based approaches for the transgenic suppression of DENV in transformed mosquito cells and tissues.

In this qualitative study, in-depth interviews were conducted with 15 surrogates from India. Two themes are discussed: (1) motivations for becoming surrogates and (2) role of family and community in surrogacy. The respondents were illiterate women aged 21–30 years and had been surrogate mothers an average of 2 times. Motivations for surrogacy were financial in nature. Surrogates reported stigma from extended family and community forcing them to leave their homes and relocate after surrogacy. This study recommends counseling and psychosocial support services to women and change in policy to execute a stringent law to protect the rights of surrogates.

IntroductionApproximately 100 million confirmed infections and 20,000 deaths are caused by Dengue virus (DENV) outbreaks annually. Global warming and rapid dispersal have resulted in DENV epidemics in formally non-endemic regions. Currently no consistently effective preventive measures for DENV exist, prompting development of transgenic and paratransgenic vector control approaches. Production of transgenic mosquitoes refractory for virus infection and/or transmission is contingent upon defining antiviral genes that have low probability for allowing escape mutations, and are equally effective against multiple serotypes. Previously we demonstrated the effectiveness of an anti-viral group I intron targeting U143 of the DENV genome in mediating trans-splicing and expression of a marker gene with the capsid coding domain. In this report we examine the effectiveness of coupling expression of ΔN Bax to trans-splicing U143 intron activity as a means of suppressing DENV infection of mosquito cells.ResultsTargeting the conserved DENV circularization sequence (CS) by U143 intron trans-splicing activity appends a 3’ exon RNA encoding ΔN Bax to the capsid coding region of the genomic RNA, resulting in a chimeric protein that induces premature cell death upon infection. TCID50-IFA analyses demonstrate an enhancement of DENV suppression for all DENV serotypes tested over the identical group I intron coupled with the non-apoptotic inducing firefly luciferase as the 3’ exon. These cumulative results confirm the increased effectiveness of this αDENV-U143-ΔN Bax group I intron as a sequence specific antiviral that should be useful for suppression of DENV in transgenic mosquitoes. Annexin V staining, caspase 3 assays, and DNA ladder observations confirm DCA-ΔN Bax fusion protein expression induces apoptotic cell death.ConclusionThis report confirms the relative effectiveness of an anti-DENV group I intron coupled to an apoptosis-inducing ΔN Bax 3’ exon that trans-splices conserved sequences of the 5’ CS region of all DENV serotypes and induces apoptotic cell death upon infection. Our results confirm coupling the targeted ribozyme capabilities of the group I intron with the generation of an apoptosis-inducing transcript increases the effectiveness of infection suppression, improving the prospects of this unique approach as a means of inducing transgenic refractoriness in mosquitoes for all serotypes of this important disease.

Plots of the frequency of occurrences of aspect angles calculated from gridded DEMs showed inordinately large spikes at the cardinal directions. Because such frequencies are not natural, the techniques of calculating slope and aspect were examined. Ultimately, the cause of the spikes was found to be the fact that the elevation values are reported only to the nearest meter. Calculations of slope and aspect angles using rounded elevation data from simulated surfaces of known slope and aspect demonstrate the nature of the problem. These simulations show that the problem of defining aspects is greatest on the most gentle slopes, as well as smaller grid spacings. Consideration was also given to various techniques of selecting elevations for deriving the parameters in the slope and aspect equations. At lower slopes, calculated aspects may be off by tens of degrees. Because there is no way to make the data more precise, researchers should exercise caution in making interpretations of aspect angles. Calculations of slope angles are affected by data precision but not as dramatically as in the calculations of aspect angles. Les tracés de la fréquence des occurences des angles d'aspect, calculés à partir de quadrillages de MNE, ont montré des pics excessivement grands vers les points cardinaux. Parce que de telles fréquences ne sont pas naturelles, on a examiné les techniques de calcul de pente et d'aspect. Finalement, on a trouvé que la présence des pics était due au fait que les valeurs d'élévation sont reportées au mètre près. Les calculs des angles de pente et d'aspect à partir de données d'élévation arrondies, provenant de surfaces de simulation de pente et d'aspect connus, démontrent bien la nature du problème. Ces simulations montrent que plus les pentes s'adoucissent et plus l'intervalle du quadrillage se rapetisse, plus le problème de définir les aspects s'accroît. On a aussi considéré diverses techniques pour choisir les élévations afin de dériver les paramètres dans les équations de pente et d'aspect. Dans les pentes les plus douces, les aspects calculés peuvent être erronés de dizaines de degrés. Parce qu'il n'est pas possible de rendre les données plus précises, les chercheurs devraient démontrer de la prudence en interprétant les angles d'aspect. Les calculs d'angles de pente sont affectés par la précision des données, mais pas aussi dramatiquement que dans les calculs d'angles d'aspect.

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Comparisons of the relative activities of 11 intergenic region (IGR) internal ribosome entry site (IRES) elements of insect dicistrovirus with 59 IRES elements of the hepatitis C and encephalomyocarditis viruses were performed in insect and mammalian cells. Dual luciferase assays were performed to determine the most effective dicistrovirus IGR IRES in the lepidopteran cell lines Sf9 (Spodoptera frugiperda) and BmN (Bombyx mori ), and the dipteran cell lines S2 (Drosophila melanogaster) and ATC-10 (Aedes aegypti ). Evaluation of dual luciferase expression from DNA plasmids and in vitro-transcribed RNA revealed apparent splicing with certain IRES elements. Though IRES activity depended upon the cell line examined, the black queen cell and Drosophila C dicistrovirus intergenic IRES elements were most effective for coupled gene expression in the diverse insect cell lines examined.The family Dicistroviridae, consisting of a single genus Cripavirus, is a newly formed family of non-enveloped, positive-sense RNA viruses that infect invertebrates (Mayo, 2002). Initially, dicistroviruses were categorized as picornalike viruses due to the morphological similarity of these viruses to members of the family Picornaviridae (e.g. poliovirus) (Mayo, 2002). In terms of overall genome organization, there are several similarities between picornaand dicistroviruses (Jan, 2006); a VPg element is present at the 59 end of the respective genomes, a 59 internal ribosome entry site (IRES) within the 59 untranslated regions (UTR) (Shibuya & Nakashima, 2006) and a poly(A) tail at the 39 end of the genome of each member of these virus families.IRES elements are cis-acting elements that function to recruit the necessary translation machinery to the desired initiator codon. These cap-independent initiators of translation tend to vary in their degree of secondary structure, GC content and requirement for the cellular proteins necessary to recruit ribosomes, thus promoting the debate over the necessity of classifying IRES elements into several classes (Baird et al., 2006). IRES elements preferentially operate during times of cellular stress, such as viral infection and DNA damage, or when cap-dependent translation initiation is compromised. These elements can be found in a wide variety of viral genomes, ranging from the retrovirus human immunodeficiency virus type-2 (Herbreteau et al., 2005) to DNA viruses such as Kaposi's sarcoma-associated herpesvirus (Bieleski & Talbot, 2001;Grundhoff & Ganem, 2001;Hellen & Sarnow, 2001), and in a wide range of cell types (Baird et al., 2006), underlining the importance of IRES elements in nature.Reported here is a comprehensive study of dicistrovirus intergenic region (IGR) IRES elements using a Renilla/ firefly (RL/FL) dual luciferase assay (Honda et al., 2000;Lerat et al., 2000). The relative activities of IGR IRES elements from 11 of the 14 currently classified dicistrovirus family members (Table 1) were compared in uninfected mammalian and insect cell lines for potential future use in expression vectors....

We have examined the effect of chronic diabetes mellitus upon cell membrane composition and turnover in streptozotocin-treated rats and control animals maintained for four to eight weeks. Liver plasma membranes, prepared from diabetic animals, showed enhanced activities of alkaline phosphatase and glucose-6-phosphatase and depressed 5'-nucleotidase when compared with controls. Studies of the nonprotein constituents of liver plasma membranes and red cell "ghosts" showed similar changes in both tissues: sialic acid and cholesterol content were reduced in the membranes of diabetic animals, while phospholipids (total and individual classes) and neutral sugars were unchanged. To look for changes in relative turnover rates of individual membrane proteins, we combined a double-label in-vivo technic using [3H] and [14C] leucine with polyacrylamide gel separation of membrane proteins. No significant differences were observed between control and diabetic animals. In chronically diabetic animals, cell membranes may show significant changes in overall composition with no significant changes in the rate of protein turnover.

BackgroundRecent epidemics of dengue viruses (DENV) coupled with new outbreaks on the horizon have renewed the demand for novel detection methods that have the ability to identify this viral pathogen prior to the manifestation of symptoms. The ability to detect DENV in a timely manner is essential for rapid recovery from disease symptoms. A modified lab-derived 10-23 DNAzyme tethered to gold nanoparticles provides a powerful tool for the detection of viruses, such as DENV.ResultsWe examined the effectiveness of coupling DNAzyme (DDZ) activation to the salt-induced aggregation of gold nanoparticles (AuNP) to detect dengue virus (DENV) progeny in mosquito cells. A DNAzyme was designed to recognize the 5’ cyclization sequence (5’ CS) that is conserved among all DENV, and conjugated to AuNPs. DDZ-AuNP has demonstrated the ability to detect the genomic RNA of our model dengue strain, DENV-2 NGC, isolated from infected Aedes albopictus C6/36 cells. These targeting events lead to the rapid aggregation of AuNPs, resulting in a red to clear color transition of the reaction mixes, and thus positive detection of the DENV RNA genome. The inclusion of SDS in the reaction mixture permitted the detection of DENV directly from cell culture supernatants without additional sample processing. Specificity assays demonstrated detection is DENV-specific, while sensitivity assays confirm detection at levels of 1 × 101 TCID50 units. These results demonstrate DDZ-AuNP effectively detects DENV genomes in a sequence specific manner and at concentrations that are practical for field use.ConclusionsWe have developed an effective detection assay using DNAzyme catalysis coupled with AuNP aggregation for the detection of DENV genomes in a sequence specific manner. Full development of our novel DDZ-AuNP detection method will provide a practical, rapid, and low cost alternative for the detection of DENV in mosquito cells and tissues, and possibly infected patient serum, in a matter of minutes with little to no specialized training required.