AXL receptor tyrosine kinase modulates gonadotropin-releasing hormone receptor signaling.

BACKGROUND: Gonadotropin-releasing hormone (GnRH) receptors are essential for reproduction and are expressed in numerous urogenital, reproductive, and non-reproductive cancers. In addition to canonical G protein-coupled receptor signaling, GnRH receptors functionally interact with several receptor tyrosine kinases. AXL is a receptor tyrosine kinase expressed in numerous tissues as well as multiple tumors. Here we tested the hypothesis that AXL, along with its endogenous ligand Gas6, impacts GnRH receptor signaling. METHODS: We used clonal murine pituitary αT3-1 and LßT2 gonadotrope cell lines to examine the effect of AXL activation on GnRH receptor-dependent signaling outcomes. ELISA and immunofluorescence were used to observe AXL and GnRH receptor expression in αT3-1 and LßT2 cells, as well as in murine and human pituitary sections. We also used ELISA to measure changes in ERK phosphorylation, pro-MMP9 production, and release of LHß. Digital droplet PCR was used to measure the abundance of Egr-1 transcripts. A transwell migration assay was used to measure αT3-1 and LßT2 migration responses to GnRH and AXL. RESULTS: We observed AXL, along with the GnRH receptor, expression in αT3-1 and LßT2 gonadotrope cell lines, as well as in murine and human pituitary sections. Consistent with a potentiating role of AXL, Gas6 enhanced GnRH-dependent ERK phosphorylation in αT3-1 and LßT2 cells. Further, and consistent with enhanced post-transcriptional GnRH receptor responses, we found that Gas6 increased the abundance of Egr-1 transcripts. Suggesting functional significance, in LßT2 cells, Gas6/AXL signaling stimulated LHß production and enhanced GnRH receptor-dependent generation of pro-MMP9 protein and promoted cell migration. CONCLUSIONS: Altogether, these data describe a novel role for AXL as a modulator of GnRH receptor signaling. Video Abstract.

An Update of Nucleic Acids Aptamers Theranostic Integration with CRISPR/Cas Technology.

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system is best known for its role in genomic editing. It has also demonstrated great potential in nucleic acid biosensing. However, the specificity limitation in CRISPR/Cas has created a hurdle for its advancement. More recently, nucleic acid aptamers known for their high affinity and specificity properties for their targets have been integrated into CRISPR/Cas systems. This review article gives a brief overview of the aptamer and CRISPR/Cas technology and provides an updated summary and discussion on how the two distinctive nucleic acid technologies are being integrated into modern diagnostic and therapeutic applications.

The Hypothalamic-Pituitary-Adrenal Axis: Development, Programming Actions of Hormones, and Maternal-Fetal Interactions.

The hypothalamic-pituitary-adrenal axis is a complex system of neuroendocrine pathways and feedback loops that function to maintain physiological homeostasis. Abnormal development of the hypothalamic-pituitary-adrenal (HPA) axis can further result in long-term alterations in neuropeptide and neurotransmitter synthesis in the central nervous system, as well as glucocorticoid hormone synthesis in the periphery. Together, these changes can potentially lead to a disruption in neuroendocrine, behavioral, autonomic, and metabolic functions in adulthood. In this review, we will discuss the regulation of the HPA axis and its development. We will also examine the maternal-fetal hypothalamic-pituitary-adrenal axis and disruption of the normal fetal environment which becomes a major risk factor for many neurodevelopmental pathologies in adulthood, such as major depressive disorder, anxiety, schizophrenia, and others.

In-Solution Molecular Recognition Comparison of Aptamers against the Herbicide Atrazine.

Atrazine is a common herbicide that is widely used to control weed growth in both agricultural and residential settings. It has been shown to act as an endocrine disruptor that affects aquatic organisms. Rapid and low-cost monitoring methods for atrazine is the first step to mitigate its widespread persistency. Aptamers are small synthetic oligonucleotides that can assume a 3D structure to act as the molecular recognition element for a specific target of interest. Two different atrazine binding aptamers (R12.23 Trunc. and R12.45 Trunc.) have been identified from the same library design but with fundamentally different in vitro selection methodologies. While the R12.23 Trunc. has been utilized in immobilized biosensing platforms, it is unclear if in-solution-based applications would be suitable for both atrazine binding aptamers. This study provides the first insight of comparative in-solution binding profiles of the two atrazine binding aptamers. Based on our results, this information will be useful for future biosensing platform development utilizing the two aptamers.

Simultaneous expression of ClopHensor and SLC26A3 reveals the nature of endogenous oxalate transport in CHO cells.

ClopHensor, a fluorescent fusion protein, is a dual function biosensor that has been utilized as a tool for the simultaneous measurement of intracellular chloride and pH in cells. ClopHensor has traditionally been used in conjunction with fluorescence microscopy for single cell measurements. Here, we present a promising multi-well format advancement for the use of ClopHensor as a potential high-throughput method capable of measuring fluorescence signal intensity across a well of confluent cells with highly reproducible results. Using this system, we gained mechanistic insight into an endogenous oxalate transporter in Chinese hamster ovary (CHO) cells expressing ClopHensor and the human chloride transporter, SLC26A3. SLC26A3, a known anion exchanger, has been proposed to play a role in colonic oxalate absorption in humans. Our attempt to study the role of SLC26A3 in oxalate transport revealed the presence of an endogenous oxalate transporter in CHO cells. This transporter was strongly inhibited by niflumate, and exhibited clear saturability. Use of ClopHensor in a multi-well cell assay allowed us to quickly demonstrate that the endogenous oxalate transporter was unable to exchange chloride for bicarbonate, unlike SLC26A3.

In Vitro Selection and Characterization of a Single-Stranded DNA Aptamer Against the Herbicide Atrazine.

Atrazine is an herbicide that is widely used in crop production at about 70 million pounds per year in the United States. Its widespread use has led to contamination of groundwater and other aquatic systems. It has resulted in many serious environmental and human health issues. This study focuses on the identification and characterization of a single-stranded DNA (ssDNA) aptamer that binds to atrazine. In this study, a variation of the systematic evolution of ligands by exponential enrichment (SELEX) process was used to identify an aptamer, which binds to atrazine with high affinity and specificity. This SELEX focused on inducing the aptamer's ability to change conformation upon binding to atrazine, and stringent negative target selections. After 12 rounds of in vitro selection, the ssDNA aptamer candidate R12.45 was chosen and truncated to obtain a 46-base sequence. The binding affinity, specificity, and structural characteristics of this truncated candidate was investigated by using isothermal titration calorimetry, circular dichroism (CD) analysis, SYBR Green I (SG) fluorescence displacement assays, and gold nanoparticles (AuNPs) colorimetric assays. The truncated R12.45 candidate aptamer bound to atrazine with high affinity (K d = 3.7 nM) and displayed low cross-binding activities on structurally related herbicides. In addition, CD analysis data indicated a target induced structural stabilization. Finally, SG assays and AuNPs assays showed nonconventional binding activities between the truncated R12.45 aptamer candidate and atrazine, which warrants future studies.