Redox cycling-based signal amplification at alkanethiol modified nanoporous gold interdigitated microelectrodes.

Interdigitated electrodes (IDEs) enable electrochemical signal enhancement through repeated reduction and oxidation of the analyte molecule. Porosity on these electrodes is often used to lower the impedance background. However, their high capacitive current and signal interferences with oxygen reduction limit electrochemical detection ability. We present utilization of alkanethiol modification on nanoporous gold (NPG) electrodes to lower their background capacitance and chemically passivate them from interferences due to oxygen reduction, while maintaining their fast electron transfer rates, as validated by lower separation between anodic and cathodic peaks (ΔE) and lower charge transfer resistance (Rct) values in comparison to planar gold electrodes. Redox amplification based on this modification enables sensitive detection of various small molecules, including pyocyanin, p-aminophenol, and selective detection of dopamine in the presence of ascorbic acid. Alkanethiol NPG arrays are applied as a multiplexed sensor testbed within a well plate to screen binding of various peptide receptors to the SARS COV2 S-protein by using a sandwich assay for conversion of PAPP (4-aminophenyl phosphate) to PAP (p-aminophenol), by the action of AP (alkaline phosphatase), which is validated against optical ELISA screens of the peptides. Such arrays are especially of interest in small volume analytical settings with complex samples, wherein optical methods are unsuitable.

Rational Approach to Optimizing Conformation-Switching Aptamers for Biosensing Applications.

The utilization of structure-switching aptamers (SSAs) has enabled the development of novel sensing platforms for the sensitive and continuous detection of molecules. De novo development of SSAs, however, is complex and laborious. Here we describe a rational approach to SSA optimization that simultaneously improves aptamer binding affinity and introduces target-dependent conformation-switching for compatibility with real-world biosensor applications. Key structural features identified from NMR and computational modeling were used to optimize conformational switching in the presence of target, while large-scale, microarray-based mutation analysis was used to map regions of the aptamer permissive to mutation and identify combinations of mutations with stronger binding affinity. Optimizations were carried out in a relevant biofluid to ensure a seamless transition of the aptamer to a biosensing platform. Initial proof-of-concept for this approach is demonstrated with a cortisol binding aptamer but can easily be translated to other relevant aptamers. Cortisol is a hormone correlated with the stress response that has been associated with various medical conditions and is present at quantifiable levels in accessible biofluids. The ability to continuously track levels of stress in real-time via cortisol monitoring, which can be enabled by the aptamers reported here, is crucial for assessing human health and performance.

Engineering a Synthetic Dopamine-Responsive Riboswitch for In Vitro Biosensing.

The detection of chemicals using natural allosteric transcription factors is a powerful strategy for point-of-use molecular sensing, particularly using fieldable cell-free gene expression (CFE) systems. However, the reliance of detection schemes on characterized protein-based sensors limits the number of measurable analytes. One alternative solution to this issue is to develop new sensors by generating RNA aptamers against the target analyte and then incorporating them directly into a riboswitch scaffold for ligand-inducible genetic control of a reporter protein. However, this strategy has not generated more than a handful of successful portable cell-free molecular sensors. To address this gap, here we convert dopamine-binding aptamers into functional dopamine-sensing riboswitches that regulate gene expression in a freeze-dried CFE reaction. We then develop an assay for direct detection and semi-quantification of dopamine in human urine. We anticipate that this work will be broadly applicable for converting many in vitro-generated RNA aptamers into fieldable molecular diagnostics.

A high-throughput pipeline for design and selection of peptides targeting the SARS-Cov-2 Spike protein.

Rapid design, screening, and characterization of biorecognition elements (BREs) is essential for the development of diagnostic tests and antiviral therapeutics needed to combat the spread of viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To address this need, we developed a high-throughput pipeline combining in silico design of a peptide library specific for SARS-CoV-2 spike (S) protein and microarray screening to identify binding sequences. Our optimized microarray platform allowed the simultaneous screening of ~ 2.5 k peptides and rapid identification of binding sequences resulting in selection of four peptides with nanomolar affinity to the SARS-CoV-2 S protein. Finally, we demonstrated the successful integration of one of the top peptides into an electrochemical sensor with a clinically relevant limit of detection for S protein in spiked saliva. Our results demonstrate the utility of this novel pipeline for the selection of peptide BREs in response to the SARS-CoV-2 pandemic, and the broader application of such a platform in response to future viral threats.

Selective and non-selective OT receptor agonists induce different locomotor behaviors in male rats via central OT receptors and peripheral V1a receptors.

Oxytocin (OT) continues to inspire much research due to its diverse physiological effects. While the best-understood actions of OT are uterine contraction and milk ejection, OT is also implicated in maternal and bonding behaviors, and potentially in CNS disorders such as autism, schizophrenia, and pain. The dissection of the mechanism of action of OT is complicated by the fact that this peptide activates not only its cognate receptor but also vasopressin type 1a (V1a) receptors. In this study, we evaluated OT and a selective OT receptor (OTR) agonist, FE 204409, in an automated assay that measures rat locomotor activity. The results showed: 1) Subcutaneous (sc) administration of OT decreased locomotor behavior (distance traveled, stereotypy, and rearing). This effect was reversed by a V1a receptor (V1aR) antagonist ([Pmp1,Tyr(ME)2]AVP, sc), suggesting that OT acts through peripheral V1aR to inhibit locomotor activity. 2) A selective OTR agonist (FE 204409, sc) increased stereotypy. This effect was reversed by an OTR antagonist dosed icv, suggesting a central OTR site of action. Our findings identify distinct behavioral effects for OT and the selective agonist FE 204409, adding to the growing body of evidence that the V1aR mediates many effects attributed to OT and that peptides administered systemically at supra-physiological doses may activate receptors in the brain. Our studies further emphasize the importance of utilizing selective agonists and antagonists to assess therapeutic indications.

Additive effect of simultaneous continuous administration of degarelix and TAK-448 on LH suppression in a castrated rat model.

Gonadotropin releasing hormone (GnRH) analogs have long been used in androgen deprivation therapy (ADT) in the treatment of prostate cancer. Chronic administration of either GnRH agonists or antagonists leads to suppression of testosterone production in the testes via either downregulation or direct blockade of the GnRH receptor in the pituitary, respectively. Chronic administration of kisspeptin analogs has more recently been shown to lead to testosterone suppression via desensitization of GnRH neurons in the hypothalamus and an optimized kisspeptin analog, TAK-448, was proven effective in a small phase 1 trial. The current study explored the hypothesis that co-administration of TAK-448 and the GnRH antagonist, degarelix, would have an additive effect on hormonal suppression, as a result of simultaneous intervention in separate steps in the same pathway. TAK-448 or degarelix were first administered individually to castrated rats in order to identify low doses capable of partial or no suppression of luteinizing hormone (LH). In the second step, combinations of the low doses of TAK-448 and degarelix were assessed in a 14 day study and compared to the drugs administered separately. The results showed that simultaneous intervention at the kisspeptin and GnRH receptors caused a more pronounced LH suppression than either drug alone, demonstrating an additive or potentiating effect. These results suggest that such a drug combination may hold promise as novel forms of androgen deprivation therapy in the treatment of prostate cancer.

Differences in academic and executive function domains among children with ADHD Predominantly Inattentive and Combined Types.

Differences between the subtypes of Attention Deficit Hyperactivity Disorder (ADHD) continue to have a place in the clinical and research literature. The purpose of this study was to examine differences specific to academic and executive function deficits in a sample of 40 children, aged 9-15 years. Although there was a tendency for the Predominantly Inattentive (PI) group to evidence lower performance on calculation and written expression tasks, these differences dissipated when IQ was included as a covariate. For executive function domains of set shifting, interference, inhibition, and planning, differences emerged for interference, but only when girls were excluded from the analysis and no control for IQ was made. For parent ratings of executive function, expected differences were found on the Inhibit scale with the Combined Type (CT) group evidencing greater problems in this area; this difference remained even when girls were excluded and IQ was controlled. Implications for research and practice are presented.

Attention Deficit Hyperactivity Disorder: manifestation in adulthood.

The purpose of the current study was to investigate the manifestation of ADHD in adults using a combination of structured clinical interview, behavioral self-report, and a range of neuropsychological measures. Symptom criteria that are endorsed by adults with ADHD as compared to non-diagnosed adults and an adult sample with other clinical disorders tend to reflect problems with follow-through, forgetting, organization, and losing things. Notably, adults in the No Diagnosis group endorsed a higher frequency of symptoms than base rates reported elsewhere. Related to sense of time, adults with ADHD endorsed problems with meeting deadlines, not completing tasks, not planning ahead, and having a poorer sense of time significantly more frequently than adults in either the No-Diagnosis or Other Clinical Disorder group. Results highlighted the need for further research specific to the manifestation of ADHD in adulthood and the development of diagnostic criteria that take into account the differences in development as well as age-related differences in contextual demands.

Wisconsin Card Sorting Test with children: a meta-analytic study of sensitivity and specificity.

More and more frequently the presence of executive function deficits appears in the research literature in conjunction with disabilities that affect children. Research has been most directed at the extent to which executive function deficits may be implicated in specific disorders such as attention deficit hyperactivity disorder (ADHD); however, deficits in executive function have been found to be typical of developmental disorders in general. The focus of this paper is to examine the extent to which one frequently used measure of executive function, the Wisconsin Card Sorting Test (WCST), demonstrates sensitivity and specificity for the identification of those executive function deficits associated with ADHD as well as its use with other developmental disorders through meta-analytic methods. Evidence of sensitivity of the WCST to dysfunction of the central nervous system is reviewed. Effect sizes calculated for all studies compared groups of children on differing variables of the WCST. The results of this meta-analysis suggest that across all of the studies, individuals with ADHD fairly consistently exhibit poorer performance as compared to individuals without clinical diagnoses on the WCST as measured by Percent Correct, Number of Categories, Total Errors, and Perseverative Errors. Notably, other various clinical groups performed more poorly than the ADHD groups in a number of studies. Thus, while impaired performance on the WCST may be indicative of an underlying neurological disorder, most likely related to frontal lobe function, poor performance is not sufficient for a diagnosis of ADHD. Implications for further research are presented.

The Tower of London and neuropsychological assessment of ADHD in adults.

Executive function refers to a variety of behaviors and abilities related to planning and strategy use, as well as the maintenance of attention and behavior in the pursuit of some goal; these behaviors are generally deficient in individuals with Attention Deficit Hyperactivity Disorder (ADHD). The Tower of London (TOL) is one task used in the assessment of executive function. For adults with ADHD, there is minimal research on the extent to which they demonstrate impaired performance on tower tasks. With a sample of 102 individuals between the ages of 16 and 33 years, the extent to which performance on the TOL-Drexel Edition (TOL(DX)) was related to performance on other measures of executive function and diagnostic grouping was investigated. Results indicated that TOL(DX) variables are not correlated significantly with age or Global Assessment of Functioning (GAF). Of the TOL(DX) variables, only Rule Violations correlated with multiple other executive function variables. Rule Violations correlated minimally, but significantly, with cognitive ability, perceptual skills, Matrix Reasoning, Processing Speed, and immediate memory. As might be expected, Processing Speed also significantly correlated with Total Time and Time Violations. Notably, scores on the TOL(DX) did not correlate significantly with behavioral self-report; no between-group (ADHD, Clinical Control, No Diagnosis) differences emerged for any of the TOL(DX) variables. Further, with this sample, mean scores across the TOL(DX) variables were well within the average range. Taken together, these results suggest that while the TOL(DX) measures aspects of ability not tapped by other measures, and may therefore provide additional information on individual functioning, results should not be interpreted as indicative of the presence or absence of a disorder.