One-step RT-qPCR assay for ZIKV RNA detection in Aedes aegypti samples: a protocol to study infection and gene expression during ZIKV infection.

BACKGROUND: Zika virus (ZIKV) is transmitted to humans during the bite of an infected mosquito. In a scenario of globalization and climate change, the frequency of outbreaks has and will increase in areas with competent vectors, revealing a need for continuous improvement of ZIKV detection tools in vector populations. A simple, rapid and sensitive assay for viral detection is quantitative reverse transcription polymerase chain reaction (qRT-PCR), yet oligos optimized for ZIKV detection in mammalian cells and samples have repeatedly shown high background when used on mosquito ribonucleic acid (RNA). In this paper, we present a one-step qRT-PCR protocol that allows for the detection of ZIKV in mosquitoes and for the evaluation of gene expression from the same mosquito sample and RNA. This assay is a less expensive qRT-PCR approach than that most frequently used in the literature and has a much lower background, allowing confident detection. METHODS: Our new oligo design to detect ZIKV RNA included in silico analysis of both viral and mosquito (Ae. aegypti and Ae. albopictus) genomes, targeting sequences conserved between Asian and African ZIKV lineages, but not matching Aedes genomes. This assay will allow researchers to avoid nonspecific amplification in insect samples due to viral integration into the mosquito genome, a phenomenon known to happen in wild and colonized populations of mosquitoes. Standard curves constructed with in vitro transcribed ZIKV RNA were used to optimize the sensitivity, efficiency and reproducibility of the assay. RESULTS: Finally, the assay was used with success to detect both ZIKV RNA in infected mosquitoes and to detect expression of the Defensin A gene, an antimicrobial peptide (AMP) involved in Aedes aegypti immune response to virus infection. CONCLUSIONS: The experimental approach to detect ZIKV RNA in Aedes aegypti presented here has demonstrated to be specific, sensitive and reliable, and additionally it allows for the analysis of mosquito gene expression during ZIKV infection.

Differential Expression of Stem Cell Markers in Human Adamantinomatous Craniopharyngioma and Pituitary Adenoma.

BACKGROUND/AIMS: Although craniopharyngioma (CP) is histologically benign, it is a pituitary tumour that grows rapidly and often recurs. Adamantinomatous CP (ACP) was associated with an activating mutation in ß-catenin, and it has been postulated that pituitary stem cells might play a role in oncogenesis in human ACP. Stem cells have also been identified in pituitary adenoma. Our aim was to characterize the expression pattern of ABCG2, CD44, DLL4, NANOG, NOTCH2, POU5F1/OCT4, SOX2, and SOX9 stem cell markers in human ACP and pituitary adenoma. METHODS AND RESULTS: We studied 33 patients (9 ACP and 24 adenoma) using real-time quantitative PCR (RT-qPCR) and immunohistochemistry. SOX9 was up-regulated in ACP, exhibiting positive immunostaining in the epithelium and stroma, with the highest expression in patients with recurrence. CD44 was overexpressed in ACP as confirmed by immunohistochemistry. SOX2 did not significantly differ among the tumour types. The RT-qPCR array showed an increased expression of MKI67,OCT4/POU5F1, and DLL4 in all tumours. NANOG was decreased in ACP. ABCG2 was down-regulated in most of the tumours. NOTCH2 was significantly decreased in the adenomas. CONCLUSION: Our results confirm the presence of stem cell markers in human pituitary tumours as well as the different expression patterns of ACP and adenoma. These findings suggest that ACP may originate from a more undifferentiated cell cluster. Additionally, SOX9 immunodetection in the stroma and the highest expression levels related to the relapse of patients suggest a contribution to the aggressive behaviour and high recurrence of this tumour type.

Differential Expression of Stem Cell Markers in Human Adamantinomatous Craniopharyngioma and Pituitary Adenoma

Background/Aims: Although craniopharyngioma (CP) is histologically benign, it is a pituitary tumour that grows rapidly and often recurs. Adamantinomatous CP (ACP) was associated with an activating mutation in ß-catenin, and it has been postulated that pituitary stem cells might play a role in oncogenesis in human ACP. Stem cells have also been identified in pituitary adenoma. Our aim was to characterize the expression pattern of ABCG2, CD44, DLL4, NANOG, NOTCH2, POU5F1/OCT4, SOX2, and SOX9 stem cell markers in human ACP and pituitary adenoma. Methods and Results: We studied 33 patients (9 ACP and 24 adenoma) using real-time quantitative PCR (RT-qPCR) and immunohistochemistry. SOX9 was up-regulated in ACP, exhibiting positive immunostaining in the epithelium and stroma, with the highest expression in patients with recurrence. CD44 was overexpressed in ACP as confirmed by immunohistochemistry. SOX2 did not significantly differ among the tumour types. The RT-qPCR array showed an increased expression of MKI67,OCT4/POU5F1, and DLL4 in all tumours. NANOG was decreased in ACP. ABCG2 was down-regulated in most of the tumours. NOTCH2 was significantly decreased in the adenomas. Conclusion: Our results confirm the presence of stem cell markers in human pituitary tumours as well as the different expression patterns of ACP and adenoma. These findings suggest that ACP may originate from a more undifferentiated cell cluster. Additionally, SOX9 immunodetection in the stroma and the highest expression levels related to the relapse of patients suggest a contribution to the aggressive behaviour and high recurrence of this tumour type.

São Paulo urban heat islands have a higher incidence of dengue than other urban areas

Urban heat islands are characterized by high land surface temperature, low humidity, and poor vegetation, and considered to favor the transmission of the mosquito-borne dengue fever that is transmitted by the Aedes aegypti mosquito. We analyzed the recorded dengue incidence in Sao Paulo city, Brazil, in 2010-2011, in terms of multiple environmental and socioeconomic variables. Geographical information systems, thermal remote sensing images, and census data were used to classify city areas according to land surface temper- ature, vegetation cover, population density, socioeconomic status, and housing standards. Of the 7415 dengue cases, a majority (93.1%) mapped to areas with land surface temperature >28 ◦ C. The dengue incidence rate (cases per 100,000 inhabitants) was low (3.2 cases) in high vegetation cover areas, but high (72.3 cases) in low vegetation cover areas where the land surface temperature was 29 ± 2 ◦ C. Interestingly, a multiple cluster analysis phenogram showed more dengue cases clustered in areas of land surface temperature >32 ◦ C, than in areas characterized as low socioeconomic zones, high population density areas, or slum-like areas. In laboratory experiments, A. aegypti mosquito larval development, blood feeding, and oviposition associated positively with temperatures of 28-32 ◦ C, indicating these temperatures to be favorable for dengue transmission. Thus, among all the variables studied, dengue incidence was most affected by the temperature.

São Paulo urban heat islands have a higher incidence of dengue than other urban areas.

Urban heat islands are characterized by high land surface temperature, low humidity, and poor vegetation, and considered to favor the transmission of the mosquito-borne dengue fever that is transmitted by the Aedes aegypti mosquito. We analyzed the recorded dengue incidence in Sao Paulo city, Brazil, in 2010-2011, in terms of multiple environmental and socioeconomic variables. Geographical information systems, thermal remote sensing images, and census data were used to classify city areas according to land surface temperature, vegetation cover, population density, socioeconomic status, and housing standards. Of the 7415 dengue cases, a majority (93.1%) mapped to areas with land surface temperature >28°C. The dengue incidence rate (cases per 100,000 inhabitants) was low (3.2 cases) in high vegetation cover areas, but high (72.3 cases) in low vegetation cover areas where the land surface temperature was 29±2°C. Interestingly, a multiple cluster analysis phenogram showed more dengue cases clustered in areas of land surface temperature >32°C, than in areas characterized as low socioeconomic zones, high population density areas, or slum-like areas. In laboratory experiments, A. aegypti mosquito larval development, blood feeding, and oviposition associated positively with temperatures of 28-32°C, indicating these temperatures to be favorable for dengue transmission. Thus, among all the variables studied, dengue incidence was most affected by the temperature.

Effects of Plasmodium gallinaceum on hemolymph physiology of Aedes aegypti during parasite development.

Insect disease vectors show diminished fecundity when infected with Plasmodium. This phenomenon has already been demonstrated in laboratory models such as Aedes aegypti, Anopheles gambiae and Anopheles stephensi. This study demonstrates several changes in physiological processes of A. aegypti occurring upon infection with Plasmodium gallinaceum, such as reduced ecdysteroid levels in hemolymph as well as altered expression patterns for genes involved in vitellogenesis, lipid transport and immune response. Furthermore, we could show that P. gallinaceum infected A. aegypti presented a reduction in reproductive fitness, accompanied by an activated innate immune response and increase in lipophorin expression, with the latter possibly representing a nutritional resource for Plasmodium sporozoites.