Modeling a Clinical Pathway for Contraception.

BACKGROUND: The Centers for Disease Control and Prevention (CDC) produced a 72-page document titled "U.S. Selective Practice Recommendations for Contraceptive Use" in 2016. This document contains the medical eligibility criteria (MEC) for contraceptive initiation or continuation based on a patient's current health status. Notations such as Business Process Model and Notation (BPMN) and Decision Model and Notation (DMN) might be useful to model such recommendations. OBJECTIVE: Our objective was to use BPMN and DMN to model and standardize the processes and decisions involved in initiating birth control according to the CDC's MEC for birth control initiation. This model could then be incorporated into an electronic health records system or other digital platform. METHODS: Medical terminology, processes, and decisions were modeled in coordination with the CDC to ensure correctness. Challenges in terminology bindings were identified and categorized. RESULTS: A model was successfully produced. Integration of clearly defined data elements proved to be the biggest challenge. CONCLUSION: BPMN and DMN have strengths and weaknesses when modeling medical processes; however, they can be used to successfully create models for clinical pathways.

In Vitro Selection of a Single-Stranded DNA Molecular Recognition Element against the Pesticide Fipronil and Sensitive Detection in River Water.

Fipronil is a commonly used insecticide that has been shown to have environmental and human health risks. The current standard methods of detection for fipronil and its metabolites, such as GC-MS, are time consuming and labor intensive. In this study, a variant of systematic evolution of ligands by exponential enrichment (SELEX), was utilized to identify the first single-stranded DNA (ssDNA) molecular recognition element (MRE) that binds to fipronil with high affinity (Kd = 48 ± 8 nM). The selected MRE displayed low cross binding activity on various environmentally relevant, structurally unrelated herbicides and pesticides, in addition to broad-spectrum binding activity on major metabolites of fipronil and a structurally similar pesticide in prepared river samples. Additionally, a proof-of-principle fluorescent detection assay was developed by using the selected ssDNA MRE as a signal-reporting element, with a limit of detection of 105 nM in a prepared river water sample.

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.

In Vitro Selection of Cancer Cell-Specific Molecular Recognition Elements from Amino Acid Libraries.

Differential cell systematic evolution of ligands by exponential enrichment (SELEX) is an in vitro selection method for obtaining molecular recognition elements (MREs) that specifically bind to individual cell types with high affinity. MREs are selected from initial large libraries of different nucleic or amino acids. This review outlines the construction of peptide and antibody fragment libraries as well as their different host types. Common methods of selection are also reviewed. Additionally, examples of cancer cell MREs are discussed, as well as their potential applications.

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.

In vitro selection of single-stranded DNA molecular recognition elements against S. aureus alpha toxin and sensitive detection in human serum.

Alpha toxin is one of the major virulence factors secreted by Staphylococcus aureus, a bacterium that is responsible for a wide variety of infections in both community and hospital settings. Due to the prevalence of S. aureus related infections and the emergence of methicillin-resistant S. aureus, rapid and accurate diagnosis of S. aureus infections is crucial in benefiting patient health outcomes. In this study, a rigorous Systematic Evolution of Ligands by Exponential Enrichment (SELEX) variant previously developed by our laboratory was utilized to select a single-stranded DNA molecular recognition element (MRE) targeting alpha toxin with high affinity and specificity. At the end of the 12-round selection, the selected MRE had an equilibrium dissociation constant (Kd) of 93.7 ± 7.0 nM. Additionally, a modified sandwich enzyme-linked immunosorbent assay (ELISA) was developed by using the selected ssDNA MRE as the toxin-capturing element and a sensitive detection of 200 nM alpha toxin in undiluted human serum samples was achieved.

In vitro selection of a single-stranded DNA molecular recognition element specific for bromacil.

Bromacil is a widely used herbicide that is known to contaminate environmental systems. Due to the hazards it presents and inefficient detection methods, it is necessary to create a rapid and efficient sensing device. Towards this end, we have utilized a stringent in vitro selection method to identify single-stranded DNA molecular recognition elements (MRE) specific for bromacil. We have identified one MRE with high affinity (K d = 9.6 nM) and specificity for bromacil compared to negative targets of selection and other pesticides. The selected ssDNA MRE will be useful as the sensing element in a field-deployable bromacil detection device.

Identification of an antibody fragment specific for androgen-dependent prostate cancer cells.

BACKGROUND: Prostate cancer is the most-diagnosed non-skin cancer among males in the US, and the second leading cause of cancer-related death. Current methods of treatment and diagnosis are not specific for the disease. This work identified an antibody fragment that binds selectively to a molecule on the surface of androgen-dependent prostate cancer cells but not benign prostatic cells. RESULTS: Antibody fragment identification was achieved using a library screening and enrichment strategy. A library of 109 yeast-displayed human non-immune antibody fragments was enriched for those that bind to androgen-dependent prostate cancer cells, but not to benign prostatic cells or purified prostate-specific membrane antigen (PSMA). Seven rounds of panning and fluorescence-activated cell sorting (FACS) screening yielded one antibody fragment identified from the enriched library. This molecule, termed HiR7.8, has a low-nanomolar equilibrium dissociation constant (Kd) and high specificity for androgen-dependent prostate cancer cells. CONCLUSIONS: Antibody fragment screening from a yeast-displayed library has yielded one molecule with high affinity and specificity. With further pre-clinical development, it is hoped that the antibody fragment identified using this screening strategy will be useful in the specific detection of prostate cancer and in targeted delivery of therapeutic agents for increased efficacy and reduced side effects.

In vitro selection of a single-stranded DNA molecular recognition element against atrazine.

Widespread use of the chlorotriazine herbicide, atrazine, has led to serious environmental and human health consequences. Current methods of detecting atrazine contamination are neither rapid nor cost-effective. In this work, atrazine-specific single-stranded DNA (ssDNA) molecular recognition elements (MRE) were isolated. We utilized a stringent Systematic Evolution of Ligands by Exponential Enrichment (SELEX) methodology that placed the greatest emphasis on what the MRE should not bind to. After twelve rounds of SELEX, an atrazine-specific MRE with high affinity was obtained. The equilibrium dissociation constant (Kd) of the ssDNA sequence is 0.62 ± 0.21 nM. It also has significant selectivity for atrazine over atrazine metabolites and other pesticides found in environmentally similar locations and concentrations. Furthermore, we have detected environmentally relevant atrazine concentrations in river water using this MRE. The strong affinity and selectivity of the selected atrazine-specific ssDNA validated the stringent SELEX methodology and identified a MRE that will be useful for rapid atrazine detection in environmental samples.

In vitro selection of a single-stranded DNA molecular recognition element for the pesticide malathion.

Many large-scale applications of the organophosphate pesticide malathion have led to widespread environmental contamination. Concentrations are found in the environment well above those which are harmful to humans and environmental organisms. No current method of detection for this pesticide is rapid, cost-effective, and specific for malathion. Therefore, we utilized a stringent Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process to identify a Molecular Recognition Element (MRE) for malathion. This MRE was identified from a large ssDNA library and has an equilibrium dissociation constant (Kd) in the low-nanomolar range. Additionally, it has significant selectivity for malathion in comparison to various other pesticides and metabolites of malathion, which were used as negative targets of selection. The high affinity and selectivity of the ssDNA MRE for malathion is a product of the stringent SELEX selection scheme and will be useful for rapid, inexpensive, and specific detection of malathion in the environment.

The effect of DNA-dispersed single-walled carbon nanotubes on the polymerase chain reaction.

The unique properties of single-wall carbon nanotubes (SWCNT) make them useful in many new technologies and applications. The interaction of DNA and SWCNT is of interest for many uses, including molecular sensors. This study determined polymerase chain reaction (PCR) efficiency in amplifying a 76 base pair DNA sequence in the presence of SWCNT, of heterogeneous "Mix" and (6,5)-enriched chiralities, associated with three DNA sequences. The dependence of PCR efficiency on the concentration of DNA:SWCNT preparations was measured, as well as their age and level of dispersion (less than one month or between four and ten months). Additionally, the ability to directly amplify the DNA sequence associated with the SWCNT scaffold was investigated. In PCRs with DNA:SWCNT preparations less than one month old, concentrations greater than or equal to 0.1 mg/mL inhibited the PCR reaction. In PCRs with older preparations, no inhibition was seen at 0.01 or 0.1 mg/mL, with amplification at 1 mg/mL in some samples. Additionally, our studies showed that the DNA directly associated with the SWCNT can be amplified using PCR. This work provides an inhibitory concentration of DNA-dispersed SWCNT in PCR reactions for different preparations as well as a basis for future DNA:SWCNT studies that require PCR amplification. This will be useful for future studies focused on the use of SWCNT in molecular sensing technologies.

Assessment of aptamer-steroid binding using stacking-enhanced capillary electrophoresis.

The binding affinity of 17ß-estradiol with an immobilized DNA aptamer was measured using capillary electrophoresis. Estradiol captured by the immobilized DNA was injected into the separation capillary using pH-mediated sample stacking. Stacked 17ß-estradiol was then separated using micellar electrokinetic capillary chromatography and detected with UV-visible absorbance. Standard addition was used to quantify the concentration of estradiol bound to the aptamer. Following incubation with immobilized DNA, analysis of free and bound estradiol yielded a dissociation constant of 70 ± 10 µM. The method was also used to screen binding affinity of the aptamer for estrone and testosterone. This study demonstrates the effectiveness of capillary electrophoresis to assess the binding affinity of DNA aptamers.

Detection of adenosine triphosphate with an aptamer biosensor based on surface-enhanced Raman scattering.

A simple, ultrasensitive, highly selective, and reagent-free aptamer-based biosensor has been developed for quantitative detection of adenosine triphosphate (ATP) using surface-enhanced Raman scattering (SERS). The sensor contains a SERS probe made of gold nanostar@Raman label@SiO(2) core-shell nanoparticles in which the Raman label (malachite green isothiocyanate, MGITC) molecules are sandwiched between a gold nanostar core and a thin silica shell. Such a SERS probe brings enhanced signal and low background fluorescence, shows good water-solubility and stability, and exhibits no sign of photobleaching. The aptamer labeled with the SERS probe is designed to hybridize with the cDNA on a gold film to form a rigid duplex DNA. In the presence of ATP, the interaction between ATP and the aptamer results in the dissociation of the duplex DNA structure and thereby removal of the SERS probe from the gold film, reducing the Raman signal. The response of the SERS biosensor varies linearly with the logarithmic ATP concentration up to 2.0 nM with a limit of detection of 12.4 pM. Our work has provided an effective method for detection of small molecules with SERS.

Reflections on a novel therapeutic candidate.

Aptamers composed of L-nucleic acids, Spiegelmers, were selected to specifically bind GnRH. Spiegelmer inhibition of GnRH activity was demonstrated in both cellular and animal models. Rabbit studies showed minimal immunogenic response to the agents.

Automated acquisition of aptamer sequences.

While the in vitro selection of nucleic acid binding species (aptamers) requires numerous liquid-handling steps, these steps are relatively straightforward and the overall process is therefore amenable to automation. Here we demonstrate that automated selection techniques are capable of generating aptamers against a number of diverse protein targets. Automated selection techniques can be integrated with automated analytical methods, including sequencing, determination of binding constants, and structural analysis. The methods that have so far been developed can be further multiplexed, and it should soon be possible to attempt the selection of aptamers against organismal proteomes or metabolomes.