Diseases transmitted by hematophagous (blood-feeding) insects are responsible for millions of human deaths worldwide. In hematophagous insects, the blood meal is important for regulating egg maturation. Although a high concentration of iron is toxic for most organisms, hematophagous insects seem unaffected by the iron load in a blood meal. One means by which hematophagous insects handle this iron load is, perhaps, by the expression of iron-binding proteins, specifically the iron storage protein ferritin. In vertebrates, ferritin is an oligomer composed of two types of subunits called heavy and light chains, and is part of the constitutive antioxidant response. Previously, we found that the insect midgut, a main site of iron load, is also a primary site of ferritin expression and that, in the yellow fever mosquito, Aedes aegypti, the expression of the ferritin heavy-chain homologue (HCH) is induced following blood feeding. We now show that the expression of the Aedes ferritin light-chain homologue (LCH) is also induced with blood-feeding, and that the genes of the LCH and HCH are tightly clustered. mRNA levels for both LCH-and HCH-genes increase with iron, H 2 O 2 and hemin treatment, and the temporal expression of the genes is very similar. These results confirm that ferritin could serve as the cytotoxic protector in mosquitoes against the oxidative challenge of the bloodmeal. Finally, although the Aedes LCH has no iron responsive element (IRE) at its 5¢-untranslated region (UTR), the 5¢-UTR contains several introns that are alternatively spliced, and this alternative splicing event is different from any ferritin message seen to date.

Infection with mucosotropic human papillomavirus (HPV) is the necessary cause of cervical intraepithelial neoplasia. Several epidemiological studies suggest that HPV viral load can be a risk factor of cervical dysplasia. The purpose of the present study was to evaluate a methodology to determine HPV viral load of eight oncogenic HPV types (16, 18, 31, 39, 45, 51, 52, and 58). The quantitation assay is based on a high-throughput real-time PCR. The E6-E7 region of HPV types 16, 18, 45, and 51 were used to amplify specific DNA sequences and cloned into a plasmid vector. The constructs for HPV types 16, 18, 45, and 51, and the whole genome for HPV types 31, 39, 52, and 58 were quantitated using a limiting dilution analysis and used to create standard curves. Type-specific HPV clones were used to determine specificity, linearity, and intra-and inter-assay variation. The sensitivity of our assay covered a dynamic range of up to seven orders of magnitude with a coefficient of intraassay variation less than 6% and the inter-assay variation less than 20%. No cross-reactivity was observed on any of the type-specific standard curves when phylogenetically close HPV types were used as templates. Among all types of human papillomavirus (ϳ100 types) the ones classified as oncogenic types are of clinical significance due to their association with cervical neoplastic development and carcinoma. Although most HPV infections are transient, a number of host and viral factors may lead to a persistent infection thus increasing the risk of developing neoplasia.1 Understanding the natural history of oncogenic HPV infection and the predictive value of one or several HPV test, is, therefore, essential for the development of appropriate cervical cancer prevention and control programs. The limitations of cytological analysis have pressured the development of alternative screening methods in population studies, with molecular diagnosis emerging as a promising diagnostic tool in cervical cancer prevention.2 Quality assurance of these emerging technologies is paramount for large-scale clinical investigations.3 In population-based studies, questions of reliability, sensitivity, specificity, reproducibility, and scalable capacity for automation are central in selecting assays to determine factors associated with cervical carcinogenesis.Prevalence studies conducted worldwide indicate that approximately 30% of women are infected with HPV. Therefore, the use of HPV testing as a primarily screening tool is not sufficient. 4 Alternative measures that may have greater predicted value, such as HPV viral load, are currently under investigation. Several studies have correlated HPV viral load and its prognostic significance in the evolution of cervical intraepithelial neoplasia. 5,6 The methodologies used to quantitate viral burden have varying levels of specificity and sensitivity and performance evaluation is frequently not comprehensive. Hybrid capture has been a common technique used to determine HPV viral load, but does not have the capacity to diffe...