Validation of heat-inducible Ixodes scapularis HSP70 and tick-specific 3xP3 promoters in ISE6 cells.

Ixodes scapularis is an important vector of many pathogens, including the causative agent of Lyme disease. The gene function studies in I. scapularis and other ticks are hampered by the lack of genetic tools, including an inducible promoter for temporal control over transgene-encoding protein or double-stranded RNA. We characterized an intergenic sequence upstream of a heat shock protein 70 (HSP70) gene that can drive Renilla luciferase and mCherry expression in the I. scapularis cell line ISE6 (IsHSP70). In another construct, we replaced the Drosophila melanogaster minimal HSP70 promoter of the 3xP3 promoter with a minimal portion of IsHSP70 promoter and generated an I. scapularis-specific 3xP3 (Is3xP3) promoter. Both IsHSP70 and Is3xP3 have a heat-inducible expression of mCherry fluorescence in ISE6 cells with an approximately 10-fold increase in the percentage of fluorescent cells upon 2 h heat shock. These promoters described will be valuable tools for gene function studies.

Arthropod promoters for genetic control of disease vectors.

Vector-borne diseases (VBDs) impose devastating effects on human health and a heavy financial burden. Malaria, Lyme disease, and dengue fever are just a few examples of VBDs that cause severe illnesses. The current strategies to control VBDs consist mainly of environmental modification and chemical use, and to a small extent, genetic approaches. The genetic approaches, including transgenesis/genome modification and gene-drive technologies, provide the basis for developing new tools for VBD prevention by suppressing vector populations or reducing their capacity to transmit pathogens. The regulatory elements such as promoters are required for a robust sex-, tissue-, and stage-specific transgene expression. As discussed in this review, information on the regulatory elements is available for mosquito vectors but is scant for other vectors.

Validation of a heat-inducible Ixodes scapularis HSP70 promoter and developing a tick-specific 3xP3 promoter sequence in ISE6 cells.

Ixodes scapularis is an important vector of many pathogens, including the causative agent of Lyme disease, tick-borne encephalitis, and anaplasmosis. The study of gene function in I. scapularis and other ticks has been hampered by the lack of genetic tools, such as an inducible promoter to permit temporal control over transgenes encoding protein or double-stranded RNA expression. Studies of vector-pathogen relationships would also benefit from the capability to activate anti-pathogen genes at different times during pathogen infection and dissemination. We have characterized an intergenic sequence upstream of the heat shock protein 70 (HSP70) gene that can drive Renilla luciferase expression and mCherry fluorescence in the I. scapularis cell line ISE6. In another construct, we replaced the Drosophila melanogaster minimal HSP70 promoter in the synthetic 3xP3 promoter with a minimal portion of the I. scapularis HSP70 promoter and generated an I. scapularis specific 3xP3 (Is3xP3) promoter. Both promoter constructs, IsHSP70 and Is3xP3, allow for heat-inducible expression of mCherry fluorescence in ISE6 cells with an approximately 10-fold increase in the percentage of fluorescent positive cells upon exposure to a 2 h heat shock. These promoters described here will be valuable tools for gene function studies and temporal control of gene expression, including anti-pathogen genes.

Increased (Pro)renin Receptor Expression in the Hypertensive Human Brain.

Overactivation of the renin-angiotensin system (RAS) - a central physiological pathway involved in controlling blood pressure (BP) - leads to hypertension. It is now well-recognized that the central nervous system (CNS) has its own local RAS, and the majority of its components are known to be expressed in the brain. In physiological and pathological states, the (pro)renin receptor (PRR), a novel component of the brain RAS, plays a key role in the formation of angiotensin II (Ang II) and also mediates Ang II-independent PRR signaling. A recent study reported that neuronal PRR activation is a novel mechanism for cardiovascular and metabolic regulation in obesity and diabetes. Expression of the PRR is increased in cardiovascular regulatory nuclei in hypertensive (HTN) animal models and plays an important role in BP regulation in the CNS. To determine the clinical significance of the brain PRR in human hypertension, we investigated whether the PRR is expressed and regulated in the paraventricular nucleus of the hypothalamus (PVN) and rostral ventrolateral medulla (RVLM) - two key cardiovascular regulatory nuclei - in postmortem brain samples of normotensive (NTN) and HTN humans. Here, we report that the PRR is expressed in neurons, but not astrocytes, of the human PVN and RVLM. Notably, PRR immunoreactivity was significantly increased in both the PVN and RVLM of HTN subjects. In addition, PVN-PRR immunoreactivity was positively correlated with systolic BP (sBP) and showed a tendency toward correlation with age but not body mass index (BMI). Collectively, our data provide clinical evidence that the PRR in the PVN and RVLM may be a key molecular player in the neural regulation of BP and cardiovascular and metabolic function in humans.