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.

Evaluation of long term retention and utilization of the situation, background, assessment, recommendation (SBAR) communication technique.

INTRODUCTION: Effective communication as part of an interprofessional team is a required standard of pharmacy education. The Situation, Background, Assessment, Recommendation (SBAR) communication technique is an evidence-based method shown to improve patient safety, and is embedded in some curricula of pharmacy and other health care professions. The aim of this study is to determine whether students can utilize the SBAR communication technique during an interprofessional skills assessment one year following initial instruction. METHODS: Students are initially trained on the SBAR technique in an interprofessional setting using the Team Strategies and Tools to Enhance Performance and Patient Safety (TeamSTEPPS) method in the fall of the second professional year. One year later, students participated in a simulated interaction with a physician as part of the pulmonary module of the pharmacotherapeutics series. Faculty evaluators noted how many and which components of the SBAR technique students used during the interaction. The simulation was run for two academic years, results of which were compared. RESULTS: There was a significant difference in the number of students who used all four components of SBAR. A significant difference also existed between the use of the "situation" and "background" components. CONCLUSION: The TeamSTEPPS method appears to be an effective method to train students on the SBAR communication technique and results in long term retention. Pharmacy programs should consider the use of the TeamSTEPPS method early in their curricula.

Recent Progress in the Identification of Aptamers Against Bacterial Origins and Their Diagnostic Applications.

Aptamers have gained an increasing role as the molecular recognition element (MRE) in diagnostic assay development, since their first conception thirty years ago. The process to screen for nucleic acid-based binding elements (aptamers) was first described in 1990 by the Gold Laboratory. In the last three decades, many aptamers have been identified for a wide array of targets. In particular, the number of reports on investigating single-stranded DNA (ssDNA) aptamer applications in biosensing and diagnostic platforms have increased significantly in recent years. This review article summarizes the recent (2015 to 2020) progress of ssDNA aptamer research on bacteria, proteins, and lipids of bacterial origins that have implications for human infections. The basic process of aptamer selection, the principles of aptamer-based biosensors, and future perspectives will also be discussed.

Akt/survivin pathway inhibition enhances the apoptotic cell death-induced by alpha-santalol in human prostate cancer cells.

We have shown previously that alpha-santalol, a major component of sandalwood oil inhibits growth of cultured prostate cancer cells in vitro by causing apoptosis, but the mechanism of cell death is not fully elucidated. The present study was undertaken to investigate the role of PI3K/Akt/survivin pathway in alpha-santalol-induced apoptosis employing cultured LNCaP and PC-3 human prostate cancer cells. Treatment of prostate cancer cells with alpha-santalol (20, 40 µM) resulted in the down regulation of survivin and p-AKT (s-473) expression and statistically significant reduction in total survivin levels as evidenced by survivin ELISA assay. Furthermore, inhibition of PI3K-Akt pathway by pharmacological inhibitor, LY294002 enhanced the apoptotic cell death induced by alpha-santalol as determined by cell viability, cellular morphology, active caspase-3 activity and expression of cleaved PARP, cleaved caspase-3 levels. In conclusion, the present study provides novel insight into the molecular circuitry of alpha-santalol-induced cell death and reveals that alpha-santalol targets Akt/Survivin pathway to induce cell death and that the cell death is increased in the presence of a known inhibitor of the pathway.

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.

A Mini-Review: Clinical Development and Potential of Aptamers for Thrombotic Events Treatment and Monitoring.

The unique opportunity for aptamer uses in thrombotic events has sparked a considerable amount of research in the area. The short half-lives of unmodified aptamers in vivo remain one of the major challenges in therapeutic aptamers. Much of the incremental successful therapeutic aptamer stories were due to modifications in the aptamer bases. This mini-review briefly summarizes the successes and challenges in the clinical development of aptamers for thrombotic events, and highlights some of the most recent developments in using aptamers for anticoagulation monitoring.

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.

Selection of Single-Stranded DNA Molecular Recognition Elements against Exotoxin A Using a Novel Decoy-SELEX Method and Sensitive Detection of Exotoxin A in Human Serum.

Exotoxin A is one of the virulence factors of Pseudomonas aeruginosa, a bacterium that can cause infections resulting in adverse health outcomes and increased burden to health care systems. Current methods of diagnosing P. aeruginosa infections are time consuming and can require significant preparation of patient samples. This study utilized a novel variation of the Systematic Evolution of Ligand by Exponential Enrichment, Decoy-SELEX, to identify an Exotoxin A specific single-stranded DNA (ssDNA) molecular recognition element (MRE). Its emphasis is on increasing stringency in directing binding toward free target of interest and at the same time decreasing binding toward negative targets. A ssDNA MRE with specificity and affinity was identified after fourteen rounds of Decoy-SELEX. Utilizing surface plasmon resonance measurements, the determined equilibrium dissociation constant (Kd ) of the MRE is between 4.2 µM and 4.5 µM, and is highly selective for Exotoxin A over negative targets. A ssDNA MRE modified sandwich enzyme-linked immunosorbent assay (ELISA) has been developed and achieved sensitive detection of Exotoxin A at nanomolar concentrations in human serum. This study has demonstrated the proof-of-principle of using a ssDNA MRE as a clinical diagnostic tool.

Single-Stranded DNA Aptamers against Pathogens and Toxins: Identification and Biosensing Applications.

Molecular recognition elements (MREs) can be short sequences of single-stranded DNA, RNA, small peptides, or antibody fragments. They can bind to user-defined targets with high affinity and specificity. There has been an increasing interest in the identification and application of nucleic acid molecular recognition elements, commonly known as aptamers, since they were first described in 1990 by the Gold and Szostak laboratories. A large number of target specific nucleic acids MREs and their applications are currently in the literature. This review first describes the general methodologies used in identifying single-stranded DNA (ssDNA) aptamers. It then summarizes advancements in the identification and biosensing application of ssDNA aptamers specific for bacteria, viruses, their associated molecules, and selected chemical toxins. Lastly, an overview of the basic principles of ssDNA aptamer-based biosensors is discussed.

In Vitro Selection of a Single-Stranded DNA Molecular Recognition Element against Clostridium difficile Toxin B and Sensitive Detection in Human Fecal Matter.

Toxin B is one of the major virulence factors of Clostridium difficile, a bacterium that is responsible for a significant number of diarrhea cases in acute care settings. Due to the prevalence of C. difficile induced diarrhea, rapid and correct diagnosis is crucial in the disease management. In this study, we have employed a stringent in vitro selection method to identify single-stranded DNA molecular recognition elements (MRE) specific for toxin B. At the end of the 12-round selection, one MRE with high affinity (K d = 47.3 nM) for toxin B was identified. The selected MRE demonstrated low cross binding activities on negative targets: bovine serum albumin, Staphylococcus aureus alpha toxin, Pseudomonas aeruginosa exotoxin A, and cholera toxin of Vibrio cholera. A modified sandwich ELISA assay was developed utilizing the selected ssDNA MRE as the antigen capturing element and achieved a sensitive detection of 50 nM of toxin B in human fecal preparations.