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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.

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.

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 (Kd = 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.

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., Summary: We expressed the fluorescent fusion protein, ClopHensor, for the first time in a multi-well format to assess for bicarbonate and chloride exchange by an endogenous oxalate transporter in CHO cells.