Directed Evolution of Aptamer Discovery Technologies.

Although antibodies are a powerful tool for molecular biology and clinical diagnostics, there are many emerging applications for which nucleic acid-based aptamers can be advantageous. However, generating high-quality aptamers with sufficient affinity and specificity for biomedical applications is a challenging feat for most research laboratories. In this Account, we describe four techniques developed in our laboratory to accelerate the discovery of high-quality aptamer reagents that can achieve robust binding even for challenging molecular targets. The first method is particle display, in which we convert solution-phase aptamers into aptamer particles that can be screened via fluorescence-activated cell sorting (FACS) to quantitatively isolate individual aptamer particles based on their affinity. This enables the efficient isolation of high-affinity aptamers in fewer selection rounds than conventional methods, thereby minimizing selection biases and reducing the emergence of artifacts in the final aptamer pool. We subsequently developed the multiparametric particle display (MPPD) method, which employs two-color FACS to isolate aptamer particles based on both affinity and specificity, yielding aptamers that exhibit excellent target binding even in complex matrixes such as serum. The third method is an alkyne-azide chemistry ("click chemistry")-based particle display (click-PD) that enables the generation and screening of "non-natural" aptamers with a wide range of base modifications. We have shown that these base-modified aptamers can achieve robust affinity and specificity for targets that have proven challenging or inaccessible with natural nucleotide-based aptamer libraries. Finally, we describe the non-natural aptamer array (N2A2) platform in which a modified benchtop sequencing instrument is used to characterize base-modified aptamers in high throughput, enabling the efficient identification of molecules with excellent affinity and specificity for their targets. This system first generates aptamer clusters on the flow-cell surface that incorporate alkyne-modified nucleobases and then performs a click reaction to couple those nucleobases to an azide-modified chemical moiety. This yields a sequence-defined array of tens of millions of base-modified sequences, which can then be characterized for affinity and specificity in a high-throughput fashion. Collectively, we believe that these advancements are helping to make aptamer technology more accessible, efficient, and robust, thereby enabling the use of these affinity reagents for a wider range of molecular recognition and detection-based applications.

Posterior medial frontal cortex regulates sympathy: A TMS study.

Harm to some elicits greater sympathy than harm to others. Here, we examine the role of posterior medial frontal cortex (PMFC) in regulating sympathy, and explore the potential role of PMFC in the related phenomena of mentalizing and representing others as connected with oneself. We down-regulated either PMFC or a control region (middle temporal visual area), then assessed feelings of sympathy for and self-other overlap with two characters described as having suffered physical harm, and who were framed as adversarial or affiliative, respectively. We also measured mentalizing performance with regard to inferring the cognitive and affective states of the adversarial character. As hypothesized, down-regulating PMFC increased sympathy for both characters. Whereas we had predicted that down-regulating PMFC would decrease mentalizing ability given the postulated role of PMFC in the mentalizing network, participants in the PMFC down-regulation condition evinced greater second-order cognitive inference ability relative to controls. We observed no effect of the TMS manipulation on self-other overlap, although sympathy and self-other overlap were positively correlated. These findings are discussed as they may inform understanding of the functional role(s) of PMFC in regulating responses broadly linked with empathy.

Treatment Thresholds for Tetranychus pacificus (Acari: Tetranychidae) in California Almonds Based on Monitoring for the Natural Enemy Scolothrips sexmaculatus (Thysanoptera: Thripidae).

Optimization of integrated pest management for Pacific spider mite, Tetranychus pacificus McGregor, in California almonds requires that miticide applications be delayed as long as possible to promote biological control, while still occurring before exponential mite increases cause defoliation. We used data from twelve untreated almond orchards between 2006 and 2019 to determine that decisions to treat T. pacificus should be made on the first monitoring date with 1.4 mites per leaf (38.1% of leaves infested) to ensure that miticide applications are made prior to reaching the treatment threshold of 5.4 per leaf an average of 4.7 d later. Modifications to this threshold based on biological control were made using capture rates of sixspotted thrips, Scolothrips sexmaculatus Pergande, on yellow adhesive traps from 14 almond orchards between 2016 and 2019. Data showed that T. pacificus density remained unchanged when there were 0.42 S. sexmaculatus per trap per week for every one T. pacificus per leaf. For orchards where T. pacificus density has exceeded action and treatment thresholds, we developed a less accurate, but more practical, model that does not require counting mites on leaves. On average, T. pacificus density remained unchanged for seven and 14 d if there were an average of five and three S. sexmaculatus per trap per week, respectively. Growers who adopt the action and treatment thresholds presented herein have the potential to avoid unnecessary miticide use, maximize reliance on biological control, and promote the long-term sustainability of California's almond industry.

Field Biology of Scolothrips sexmaculatus (Thysanoptera: Thripidae) as a Predator of Tetranychus pacificus (Acari: Tetranychidae) in California Almonds.

Biological control of tetranychid spider mites in California almonds has predominantly been associated with predatory mites in the family Phytoseiidae. However, changes in production practices, including shifts to more sustainable chemical control programs, have resulted in a trophic shift leading to the predominance of six-spotted thrips, Scolothrips sexmaculatus Pergande, as the primary mite predator. Over a 3-yr period, we studied S. sexmaculatus biology and relationship to Tetranychus pacificus McGregor in almond orchards in the southern San Joaquin Valley of California. Scolothrips sexmaculatus had two periods of activity in response to increases in T. pacificus density, one in the spring and one in the summer. Sex ratios were consistently skewed to more than 90% females captured on yellow sticky traps, with females producing eggs from May to October. Female thrips were larger than males, and both sexes were larger from June to September when temperatures were warmer and food was more abundant. In response to spider mite outbreaks, S. sexmaculatus increased from approximately 2/trap/wk to an average of 1,272/trap/wk over a 5-wk period, representing a population doubling time of 4.0 d. Over this time, we described trends in T. pacificus and S. sexmaculatus density that are consistent with population regulation through predator-prey relationships by a specialist predator.

Management of Navel Orangeworm (Lepidoptera: Pyralidae) Using Four Commercial Mating Disruption Systems in California Almonds.

The navel orangeworm, Amyelois transitella (Walker), is the most significant pest of California almonds. Direct feeding on the kernel by the larvae causes reductions in salable crop, crop quality, and exportability. Pheromone mating disruption (MD) targeting navel orangeworm is a relatively new technique with the potential to improve management. In 2017, we used replicated ~16-ha plots to compare the efficacy of four commercial MD systems (CheckMate, Cidetrak, Isomate, and Semios) for their relative impacts on the number of navel orangeworm in monitoring traps and crop quality. From 2017 to 2018, we conducted nine direct comparison studies in 16 to 40 ha almond orchards to compare conventional pest management programs to programs incorporating pheromone MD systems. Across all studies, MD reduced male moth captures in pheromone traps by >94%. In the efficacy study, use of mating disruption led to 35% and 53% reductions in kernel damage in Nonpareil and pollinizer cultivars, respectively, and an average increase in crop value of $370 ha-1. In the direct comparison, kernel damage to Nonpareil and pollinizer cultivars was reduced by 65% and 78%, respectively, resulting in an average increase in crop value of $357 ha-1. Economic analyses showed that increases in crop returns exceeded the costs of implementing MD systems with the break-even point ranging from 0.86 to 1.06% of kernel damage. These results suggest that adding MD to an existing navel orangeworm management program is a cost-effective way to reduce damage while promoting sustainable pest management practices.

Evaluation of Sticky Traps for Monitoring Scolothrips sexmaculatus (Thysanoptera: Thripidae) and Stethorus punctum (Coleoptera: Coccinellidae) as Predators of Spider Mites in California Almonds.

Changes in almond production practices have caused sixspotted thrips, Scolothrips sexmaculatus Pergande, and the coccinellid beetle, Stethorus punctum LeConte, to replace phytoseiid mites as the dominant predators of spider mites in California almonds. We conducted a series of field studies to evaluate nine commercially available adhesive traps for S. sexmaculatus and S. punctum and found that the yellow strip trap was the most effective, least expensive, and easy to use. At peak population levels, individual cards routinely caught >1,000 S. sexmaculatus and >100 S. punctum trap-1 week-1. We documented that larger traps collected more S. sexmaculatus, and more S. sexmaculatus per square area, suggesting that the trap surface was attractive. We determined the number of traps needed to have 50, 70, and 90% confidence that the averages of trap captures were within 10, 30, and 50% of the population mean. Two yellow strip traps per orchard provided 90% confidence that trap captures were within 50% of the population mean if there were an average of 16.0 S. sexmaculatus trap-1 week-1 or 7.9 S. punctum trap-1 week-1. Populations required to attain the same levels of confidence using four traps per orchard were 3.9 S. sexmaculatus and 2.9 S. punctum trap-1 week-1. We conclude that use of the yellow strip trap to monitor for natural enemies, in combination with leaf samples for spider mites, has the potential to improve integrated pest management programs for spider mites, and assist future research to understand the biology and phenology of both predator species.

Posterior medial frontal cortex and threat-enhanced religious belief: a replication and extension.

Research indicates that the posterior medial frontal cortex (pMFC) functions as a 'neural alarm' complex broadly involved in registering threats and helping to muster relevant responses. Holbrook and colleagues investigated whether pMFC similarly mediates ideological threat responses, finding that downregulating pMFC via transcranial magnetic stimulation (TMS) caused (i) less avowed religious belief despite being reminded of death and (ii) less group bias despite encountering a sharp critique of the national in-group. While suggestive, these findings were limited by the absence of a non-threat comparison condition and reliance on sham rather than control TMS. Here, in a pre-registered replication and extension, we downregulated pMFC or a control region (MT/V5) and then primed participants with either a reminder of death or a threat-neutral topic. As mentioned previously, participants reminded of death reported less religious belief when pMFC was downregulated. No such effect of pMFC downregulation was observed in the neutral condition, consistent with construing pMFC as monitoring for salient threats (e.g. death) and helping to recruit ideological responses (e.g. enhanced religious belief). However, no effect of downregulating pMFC on group bias was observed, possibly due to reliance on a collegiate in-group framing rather than a national framing as in the prior study.

Click-Particle Display for Base-Modified Aptamer Discovery.

Base-modified aptamers that incorporate non-natural chemical moieties can achieve greatly improved affinity and specificity relative to natural DNA or RNA aptamers. However, conventional methods for generating base-modified aptamers require considerable expertise and resources. In this work, we have accelerated and generalized the process of generating base-modified aptamers by combining a click-chemistry strategy with a fluorescence-activated cell sorting (FACS)-based screening methodology that measures the affinity and specificity of individual aptamers at a throughput of ∼107 per hour. Our "click-particle display (PD)" strategy offers many advantages. First, almost any chemical modification can be introduced with a commercially available polymerase. Second, click-PD can screen vast numbers of individual aptamers on the basis of quantitative on- and off-target binding measurements to simultaneously achieve high affinity and specificity. Finally, the increasing availability of FACS instrumentation in academia and industry allows for easy adoption of click-PD in a broader scientific community. Using click-PD, we generated a boronic acid-modified aptamer with ∼1 µM affinity for epinephrine, a target for which no aptamer has been reported to date. We subsequently generated a mannose-modified aptamer with nanomolar affinity for the lectin concanavalin A (Con A). The strong affinity of both aptamers is fundamentally dependent upon the presence of chemical modifications, and we show that their removal essentially eliminates aptamer binding. Importantly, our Con A aptamer exhibited exceptional specificity, with minimal binding to other structurally similar lectins. Finally, we show that our aptamer has remarkable biological activity. Indeed, this aptamer is the most potent inhibitor of Con A-mediated hemagglutination reported to date.

Affordance Compatibility Effect for Word Learning in Virtual Reality.

Rich sensorimotor interaction facilitates language learning and is presumed to ground conceptual representations. Yet empirical support for early stages of embodied word learning is currently lacking. Finding evidence that sensorimotor interaction shapes learned linguistic representations would provide crucial support for embodied language theories. We developed a gamified word learning experiment in virtual reality in which participants learned the names of six novel objects by grasping and manipulating objects with either their left or right hand. Participants then completed a word-color match task in which they were tested on the same six words and objects. Participants were faster to respond to stimuli in the match task when the response hand was compatible with the hand used to interact with the named object, an effect we refer to as affordance compatibility. In two follow up experiments, we found that merely observing virtual hands interact with the objects was sufficient to acquire a smaller affordance compatibility effect, and we found that the compatibility effect was driven primarily by responses with a compatible hand and not by responses in a compatible spatial location. Our results support theoretical views of language which ground word representations in sensorimotor experiences, and they suggest promising future routes to explore the sensorimotor foundations of higher cognition through immersive virtual experiments.

Direct Selection Strategy for Isolating Aptamers with pH-Sensitive Binding Activity.

An aptamer reagent that can switch its binding affinity in a pH-responsive manner would be highly valuable for many biomedical applications including imaging and drug delivery. Unfortunately, the discovery of such aptamers is difficult and only a few have been reported to date. Here we report the first experimental strategy for generating pH-responsive aptamers through direct selection. As an exemplar, we report streptavidin-binding aptamers that retain nanomolar affinity at pH 7.4 but exhibit a ∼100-fold decrease in affinity at pH 5.2. These aptamers were generated by incorporating a known streptavidin-binding DNA motif into an aptamer library and performing FACS-based screening at multiple pH conditions. Upon structural analysis, we found that one aptamer's affinity-switching behavior is driven by a noncanonical G-A base-pair that controls its folding in a highly pH-dependent manner. We believe our strategy could be readily extended to other aptamer-target systems because it does not require a priori structural knowledge of the aptamer or the target.

Multimodal Music Perception Engages Motor Prediction: A TMS Study.

Corticospinal excitability (CSE) in humans measured with Transcranial Magnetic Stimulation (TMS) is generally increased by the perception of other people's actions. This perception can be unimodal (visual or auditory) or multimodal (visual and auditory). The increase in TMS-measured CSE is typically prominent for muscles involved in the perceived action (muscle specificity). There are two main classes of accounts for this phenomenon. One suggests that the motor system mirrors the actions that the observer perceives (the resonance account). The other suggests that the motor system predicts the actions that the observer perceives (the predictive account). To test these accounts (which need not be mutually exclusive), subjects were presented with four versions of three-note piano sequences: sound only, sight only, audiovisual, and audiovisual with sound lagging behind (the prediction violation condition). CSE was measured in two hand muscles used to play the notes. CSE increased reliably in one muscle only for the prediction violation condition, in line with the predictive account, while the other muscle demonstrated CSE increase for all conditions, in line with the resonance account. This finding supports both predictive coding accounts as well as resonance accounts of motor facilitation during action perception.

Recruitment of the motor system during music listening: An ALE meta-analysis of fMRI data.

Several neuroimaging studies have shown that listening to music activates brain regions that reside in the motor system, even when there is no overt movement. However, many of these studies report the activation of varying motor system areas that include the primary motor cortex, supplementary motor area, dorsal and ventral pre-motor areas and parietal regions. In order to examine what specific roles are played by various motor regions during music perception, we used activation likelihood estimation (ALE) to conduct a meta-analysis of neuroimaging literature on passive music listening. After extensive search of the literature, 42 studies were analyzed resulting in a total of 386 unique subjects contributing 694 activation foci in total. As suspected, auditory activations were found in the bilateral superior temporal gyrus, transverse temporal gyrus, insula, pyramis, bilateral precentral gyrus, and bilateral medial frontal gyrus. We also saw the widespread activation of motor networks including left and right lateral premotor cortex, right primary motor cortex, and the left cerebellum. These results suggest a central role of the motor system in music and rhythm perception. We discuss these findings in the context of the Action Simulation for Auditory Prediction (ASAP) model and other predictive coding accounts of brain function.

Continuous Theta Burst Stimulation of the Posterior Medial Frontal Cortex to Experimentally Reduce Ideological Threat Responses.

Decades of behavioral science research have documented functional shifts in attitudes and ideological adherence in response to various challenges, but little work to date has illuminated the neural mechanisms underlying these dynamics. This paper describes how continuous theta burst transcranial magnetic stimulation may be employed to experimentally assess the causal contribution of cortical regions to threat-related ideological shifts. In the example protocol provided here, participants are exposed to a threat prime-an explicit reminder of their own inevitable death and bodily decomposition-following a downregulation of the posterior medial frontal cortex (pMFC) or a sham stimulation. Next, disguised within a series of distracter tasks, participants' relative degree of ideological adherence is assessed-in the present example, with regard to coalitional prejudice and religious belief. Participants for whom the pMFC has been downregulated exhibit less coalitionally biased responses to an immigrant critical of the participants' national in-group, and less conviction in positive afterlife beliefs (i.e., God, angels, and heaven), despite having recently been reminded of death. These results complement prior findings that continuous theta burst stimulation of the pMFC influences social conformity and sharing and illustrate the feasibility of investigating the neural basis of high-level social cognitive shifts using transcranial magnetic stimulation.

Site-specific incorporation of quadricyclane into a protein and photocleavage of the quadricyclane ligation adduct.

The quadricyclane (QC) ligation is a bioorthogonal reaction between a quadricyclane moiety and a nickel bis(dithiolene) derivative. Here we show that a QC amino acid can be incorporated into a protein site-specifically using the pyrrolysine-based genetic code expansion platform, and subsequently used for ligation chemistry. Additionally, we exploited the photolability of the QC ligation product to render the adduct cleavable with a handheld UV lamp. We further developed a protein purification method that involves QC ligation of biotin to a protein of interest, capture on streptavidin resin, and finally release using only UV light. The QC ligation thus brings novel chemical manipulations to the realm of bioorthogonal chemistry.

Corticospinal excitability during the processing of handwritten and typed words and non-words.

A number of studies have suggested that perception of actions is accompanied by motor simulation of those actions. To further explore this proposal, we applied Transcranial magnetic stimulation (TMS) to the left primary motor cortex during the observation of handwritten and typed language stimuli, including words and non-word consonant clusters. We recorded motor-evoked potentials (MEPs) from the right first dorsal interosseous (FDI) muscle to measure cortico-spinal excitability during written text perception. We observed a facilitation in MEPs for handwritten stimuli, regardless of whether the stimuli were words or non-words, suggesting potential motor simulation during observation. We did not observe a similar facilitation for the typed stimuli, suggesting that motor simulation was not occurring during observation of typed text. By demonstrating potential simulation of written language text during observation, these findings add to a growing literature suggesting that the motor system plays a strong role in the perception of written language.

Patient-centered interviewing is associated with decreased responses to painful stimuli: an initial fMRI study.

OBJECTIVE: To identify the functional magnetic resonance imaging (fMRI) changes associated with a patient-centered interview (PCI) and a positive provider-patient relationship (PPR). METHODS: Nine female patients participated, five randomly selected to undergo a replicable, evidence-based PCI, the other four receiving standard clinician-centered interviews (CCI). To verify that PCI differed from CCI, we rated the interviews and administered a patient satisfaction with the provider-patient relationship (PPR) questionnaire. Patients were then scanned as they received painful stimulation while viewing pictures of the interviewing doctor and control images (unknown doctor). RESULTS: Interview ratings and questionnaire results confirmed that PCIs and CCIs were performed as planned and PCIs led to a much more positive PPR. We found significantly reduced pain-related neural activation in the left anterior insula region in the PCI group when the interviewing doctor's picture was shown. CONCLUSION: This study identifies an association between a PCI that produced a positive PPR and reduced pain-related neural responses in the anterior insula. This is an initial step in understanding the neural underpinnings of a PCI. PRACTICE IMPLICATIONS: If confirmed, our results indicate one neurobiological underpinning of an effective PCI, providing an additional scientific rationale for its use clinically.

Reactivity of biarylazacyclooctynones in copper-free click chemistry.

The 1,3-dipolar cycloaddition of cyclooctynes with azides, also called "copper-free click chemistry", is a bioorthogonal reaction with widespread applications in biological discovery. The kinetics of this reaction are of paramount importance for studies of dynamic processes, particularly in living subjects. Here we performed a systematic analysis of the effects of strain and electronics on the reactivity of cyclooctynes with azides through both experimental measurements and computational studies using a density functional theory (DFT) distortion/interaction transition state model. In particular, we focused on biarylazacyclooctynone (BARAC) because it reacts with azides faster than any other reported cyclooctyne and its modular synthesis facilitated rapid access to analogues. We found that substituents on BARAC's aryl rings can alter the calculated transition state interaction energy of the cycloaddition through electronic effects or the calculated distortion energy through steric effects. Experimental data confirmed that electronic perturbation of BARAC's aryl rings has a modest effect on reaction rate, whereas steric hindrance in the transition state can significantly retard the reaction. Drawing on these results, we analyzed the relationship between alkyne bond angles, which we determined using X-ray crystallography, and reactivity, quantified by experimental second-order rate constants, for a range of cyclooctynes. Our results suggest a correlation between decreased alkyne bond angle and increased cyclooctyne reactivity. Finally, we obtained structural and computational data that revealed the relationship between the conformation of BARAC's central lactam and compound reactivity. Collectively, these results indicate that the distortion/interaction model combined with bond angle analysis will enable predictions of cyclooctyne reactivity and the rational design of new reagents for copper-free click chemistry.