Artificial chromosomes: ideal vectors?

Artificial chromosomes are DNA molecules of predictable structure, which are assembled in vitro from defined constituents that behave with the properties of natural chromosomes. Artificial chromosomes were first assembled in budding yeast and have since been useful in many aspects of yeast genetics. Several attempts have been made at building artificial chromosomes in mammals, although these have been met with limited success. Consequently, mini-chromosomes of defined structure have been developed to address questions regarding mammalian chromosome function and for biotechnological applications. Here we review progress in these areas and consider how it influences plans to build artificial chromosomes in plants and parasites.     

Does Trypanosoma cruzi stress its vectors?

Trypanosoma cruzi seems to compete with its vector, Triatoma infestans, for nutrients. When starved the resistance of infected Triatoma is reduced but it is rarely affected i f it is given an adequate food supply. Infected individuals also might be more sensitive than uninfected bugs to other environmental stress factors. Günter Schaub believes that the theory, that T. cruzi does not affect its vectors, should be re-examined.     

Are migratory waterfowl vectors of seagrass pathogens?

Migratory waterfowl vector plant seeds and other tissues, but little attention has focused on the potential of avian vectoring of plant pathogens. Extensive meadows of eelgrass (Zostera marina) in southwest Alaska support hundreds of thousands of waterfowl during fall migration and may be susceptible to plant pathogens. We recovered DNA of organisms pathogenic to eelgrass from environmental samples and in the cloacal contents of eight of nine waterfowl species that annually migrate along the Pacific coast of North America and Asia. Coupled with a signal of asymmetrical gene flow of eelgrass running counter to that expected from oceanic and coastal currents between Large Marine Ecosystems, this evidence suggests waterfowl are vectors of eelgrass pathogens.     

Towards β-globin gene-targeting with integrase-defective lentiviral vectors

We have developed an integrase-defective lentiviral (LV) vector in combination with a gene-targeting approach for gene therapy of β-thalassemia. The β-globin gene-targeting construct has two homologous stems including sequence upstream and downstream of the β-globin gene, a β-globin gene positioned between hygromycin and neomycin resistant genes and a herpes simplex virus type 1 thymidine kinase (HSVtk) suicide gene. Utilization of integrase-defective LV as a vector for the β-globin gene increased the number of selected clones relative to non-viral methods. This method represents an important step toward the ultimate goal of a clinical gene therapy for β-thalassemia.     

Do viruses use vectors to penetrate mucus barriers?

I propose a mechanism by which viruses successfully infect new individuals, despite being immotile particles with no ability for directed movement. Within cells, viral particle movements are directed by motors and elements of the cytoskeleton, but how viruses cross extracellular barriers, like mucus, remains a mystery. I propose that viruses cross these barriers by hitch-hiking on bacteria or sperm cells which can transport themselves across mucosal layers designed to protect the underlying cells from pathogen attack. An important implication of this hypothesis is that agents that block interactions between viruses and bacteria or sperm may be new tools for disease prevention.     

Mosquitoes and transmission of malaria parasites – not just vectors

The regional malaria epidemics of the early 1900s provided the basis for much of our current understanding of malaria epidemiology. Colonel Gill, an eminent malariologist of that time, suggested that the explosive nature of the regional epidemics was due to a sudden increased infectiousness of the adult population. His pertinent observations underlying this suggestion have, however, gone unheeded. Here, the literature on Plasmodium seasonal behaviour is reviewed and three historical data sets, concerning seasonal transmission of Plasmodium falciparum, are examined. It is proposed that the dramatic seasonal increase in the density of uninfected mosquito bites results in an increased infectiousness of the human reservoir of infection and, therefore, plays a key role in "kick-starting" malaria parasite transmission.     

Optimized compatible set of BioBrick™ vectors for metabolic pathway engineering

The BioBrick™ paradigm for the assembly of enzymatic pathways is being adopted and becoming a standard practice in microbial engineering. We present a strategy to adapt the BioBrick™ paradigm to allow the quick assembly of multi-gene pathways into a number of vectors as well as for the quick mobilization of any cloned gene into vectors with different features for gene expression and protein purification. A primary BioBrick™ (BB-eGFP) was developed where the promoter/RBS, multiple cloning sites, optional protein purification affinity tags and reporter gene were all separated into discrete regions by additional restriction enzymes. This primary BB-eGFP then served as the template for additional BioBrick™ vectors with different origins of replication, antibiotic resistances, inducible promoters (arabinose, IPTG or anhydrotetracycline), N- or C-terminal Histidine tags with thrombin cleavage, a LacZα reporter gene and an additional origin of mobility (oriT). All developed BioBricks™ and BioBrick™ compatible vectors were shown to be functional by measuring reporter gene expression. Lastly, a C₃₀ carotenoid pathway was assembled as a model enzymatic pathway to demonstrate in vivo functionality and compatibility of this engineered vector system.     

Insecticide exposure impacts vector–parasite interactions in insecticide-resistant malaria vectors

Currently, there is a strong trend towards increasing insecticide-based vector control coverage in malaria endemic countries. The ecological consequence of insecticide applications has been mainly studied regarding the selection of resistance mechanisms; however, little is known about their impact on vector competence in mosquitoes responsible for malaria transmission. As they have limited toxicity to mosquitoes owing to the selection of resistance mechanisms, insecticides may also interact with pathogens developing in mosquitoes. In this study, we explored the impact of insecticide exposure on Plasmodium falciparum development in insecticide-resistant colonies of Anopheles gambiae s.s., homozygous for the ace-1 G119S mutation (Acerkis) or the kdr L1014F mutation (Kdrkis). Exposure to bendiocarb insecticide reduced the prevalence and intensity of P. falciparum oocysts developing in the infected midgut of the Acerkis strain, whereas exposure to dichlorodiphenyltrichloroethane reduced only the prevalence of P. falciparum infection in the Kdrkis strain. Thus, insecticide resistance leads to a selective pressure of insecticides on Plasmodium parasites, providing, to our knowledge, the first evidence of genotype by environment interactions on vector competence in a natural Anopheles–Plasmodium combination. Insecticide applications would affect the transmission of malaria in spite of resistance and would reduce to some degree the impact of insecticide resistance on malaria control interventions.     

Construction of two recombination yeast two-hybrid vectors by in vitro recombination

Recombination-based restrictionless, ligation-independent cloning has been proven to be advantageous over restriction digestion and ligation cloning. To utilize the recombination cloning and previously constructed two-hybrid cDNA libraries, a new Gateway yeast two-hybrid bait vector, pEZY202, and a new prey vector, pEZY45, were constructed. The two-hybrid vectors were generated by in vitro recombination using a protocol that can be easily adapted for the conversion of other existing vectors. The new vectors were used to assay the interaction between the WW domain of PQBP1 (PQBPww) and the WW domain binding protein WBP11. Both PQBPww and WBP11 were cloned into a Gateway donor vector by in vitro recombination. They were then subcloned into pEZY45 and pEZY202, respectively, by in vitro recombination. The binding between PQBPww and WBP11 was reported in a two-hybrid experiment using the new vectors. The results of testing the new vectors in combination with the original vectors indicated that the new bait vector could be used to screen cDNA libraries that are constructed using the original prey vectors.     

Adeno-associated virus vectors for gene therapy: more pros than cons?

Gene therapy vectors based on the adeno-associated virus (AAV) are being developed for a widening variety of therapeutic applications. Enthusiasm for AAV is due, not only to the relative safety of these vectors, but also to advances in understanding of the unique biology of this virus. This review examines a number of long-standing concerns regarding the utility of AAV for gene transfer in light of many new insights into the biology, immunology and production of AAV.     

Are there effective resistance management strategies for vectors of human disease?*

The rotation of insecticides used by the Onchocerciasis Control Programme in West Africa is reviewed and the motives for this rotation are shown to be not only management of temephos resistance in the Simulium vectors but also constraints on what compounds are usable at particular seasons. A computer model indicates that without these seasonal constraints there is unlikely to be an advantage in a pre-planned rotation of insecticides, as compared with the prompt switching of compounds as dictated by detection of build up of resistance and switching back to the original compound if the regression of resistance is found to give the opportunity to do so. The latter sequence of events can hardly be called a ‘strategy’ for resistance management, but is what any well-managed pest control programme would be expected to do. The use of an insecticide mixture is different in principle from the use of rotation and depends on the idea that if the mixture is used from the outset, when resistance to both components of the mixture is likely to be rare, the double resistance combination would be so rare as to be dwarfed in numbers by those insects which avoid exposure altogether. The prospects for successful use of a mixture depend on each component killing a very high percentage of the exposed insects which are genetically susceptible to it. Whether this condition is met could be tested, for example, in the case of exposure of mosquitoes to insecticide-treated bednets.     

Synthesis of 17α-substituted ethynylestradiols: Potential ligands for drug vectors

17α-substituted ethynylestradiols, derived from estrone, were converted to their corresponding 17α-(bromo- or iodo-propargyl)estrone intermediates. Nucleophilic substitution onto these moieties with malonate diester followed by hydrolysis and complexation with cis-Pt(Me₂en)I₂ (Me₂en = N,N-dimethylethylenediamine) gave cis-Pt(Me₂en)(2-(3-(17β-estradiol-17α-yl)-prop-2-ynyl)malonato) 7, thus demonstrating that these estrogen-derived compounds can be used to synthesize stable Pt(II) complexes. The 3-(17β-estradiol-17α-yl)-prop-2-ynyl-1-sulfanylethylthiol 23 was also prepared.     

Infection prevalence and vector-borne transmission: are vectors always to blame?

The potential for vector-independent transmission of pathogens to occur in what is generally considered to be a vector-borne system is a subject that has received little direct attention. The circumstances under which such a process might take place could conceivably be described as 'occasional' under natural conditions and 'accidental' under unnatural conditions. A more immediate concern is the ability to detect the presence of vector-independent transmission in action and, where possible, to quantify its contribution to overall infection prevalence. As intrinsically difficult as this process might be, careful observation and the use of laboratory and field-scale experiments have indicated that alternative, vector-independent routes of transmission do exist and might contribute significantly to overall prevalence in some host-vector-pathogen systems.     

Solitary bees – Potential vectors for gene flow from cultivated to wild strawberries

The genus Fragaria (Rosaceae) contains 24 plant species, including hybrid species such as the octoploid garden strawberry (F. × ananassa). Natural hybridization between Fragaria species has repeatedly been reported, and the potential future cultivation of genetically modified strawberries has made the study of hybridization potential between F. × ananassa and its wild relatives increasingly important. In Europe, F. × ananassa is the only octoploid species present, and the most likely candidate for hybridization is the common diploid woodland strawberry (F. vesca). To date, it is unknown whether pollinator spectra of the two Fragaria species overlap and thus might promote interspecific gene flow. We carried out a survey of flower visitors in northwestern Switzerland to identify major flower visitors of F. vesca and F. × ananassa. This survey indicated that wild bees are the most important shared flower visitors of F. × ananassa and F. vesca. Therefore, we studied flower choice behavior of the common wild bee Osmia bicornis in a greenhouse experiment. Osmia bicornis did not discriminate between F. × ananassa and F. vesca flowers. We conclude that wild bees are important shared flower visitors of both F. × ananassa and F. vesca and are potential vectors for gene flow between cultivated and wild strawberries.     

Double-liposome - based dual-drug delivery system as vectors for effective management of peptic ulcer

The aim of the present investigation was to prepare and evaluate a vesicular dual-drug delivery system for effective management of the mucosal ulcer. Inner encapsulating and double liposomes were prepared by the glass-bead and reverse-phase evaporation methods, respectively. The formulation consisted of inner liposomes bearing ranitidine bismuth citrate (RBC) and outer liposomes encapsulating amoxicillin trihydrate (AMOX). The optimized inner liposomes and double liposomes were extensively characterized for vesicle size, morphology, zeta potential, vesicles count, entrapment efficiency, and in vitro drug release. In vitro, the double liposomes demonstrated a sustained release of AMOX and RBC of 93.6 ± 1.9 and 84.1 ± 0.9%, respectively, at the end of 144 hours. Ex vivo studies were conducted on Helicobacter pylori (ATCC26695) bacterial cell lines. Double liposomes showed a more enhanced percent H. pylori growth inhibition than the plain drug combination. Further, in vivo studies illustrated enhanced antisecretory and ulcer-protective activity of double liposomes, as compared to the plain drug combination. Microscopic studies also supported the ulcer-protective action of the formulation. Thus, it may be concluded that double liposomes are instrumental in reducing gastric secretions and targeting ulcer sites with the interception of minimal side effects, thus suggesting their potential in ulcer therapy.