Rapid and Scalable Characterization of CRISPR Technologies Using an E. coli Cell-Free Transcription-Translation System.

CRISPR-Cas systems offer versatile technologies for genome engineering, yet their implementation has been outpaced by ongoing discoveries of new Cas nucleases and anti-CRISPR proteins. Here, we present the use of E. coli cell-free transcription-translation (TXTL) systems to vastly improve the speed and scalability of CRISPR characterization and validation. TXTL can express active CRISPR machinery from added plasmids and linear DNA, and TXTL can output quantitative dynamics of DNA cleavage and gene repression-all without protein purification or live cells. We used TXTL to measure the dynamics of DNA cleavage and gene repression for single- and multi-effector CRISPR nucleases, predict gene repression strength in E. coli, determine the specificities of 24 diverse anti-CRISPR proteins, and develop a fast and scalable screen for protospacer-adjacent motifs that was successfully applied to five uncharacterized Cpf1 nucleases. These examples underscore how TXTL can facilitate the characterization and application of CRISPR technologies across their many uses.

Attentional adjustment in priming tasks: control strategies depend on context.

Goal-directed behavior is assumed to require processes of attentional biasing to counter unwanted action tendencies elicited by distracting stimulus information. This is particularly so if stimulus categories that define the target and the distractor frequently reverse, requiring participants to respond to previously ignored stimulus categories and vice versa. In the current study, we investigated control strategies under such conditions. Specifically, we assessed trial-to-trial modulation of distractor-interference (i.e., congruency sequence effect, CSE) in a temporal flanker task associated with repetition versus alternation of the assignment of stimulus category (i.e., digits, letters) to targets and distractors (i.e., the character presented second or first, respectively) under conditions of a long SOA of 1000 ms (Experiment 1A) and 1200 ms (Experiment 1B). Whereas previous research, using a shorter SOA, suggested temporal-order control (i.e., the occurrence of a CSE in both repetition and-albeit less pronounced-alternation trials), lengthening the distractor-target SOA resulted in a CSE confined to repetition trials, suggesting strong or exclusive reliance on stimulus categories for attentional control (Experiment 1A and B). Adding a redundant stimulus feature (i.e., color), discriminating targets and distractors, eliminated the difference of CSE patterns in repetition and alternation trials (Experiment 2). Together, our results suggest that the strength of concurrently applied control strategies or the choice of a particular control strategy depend on contextual factors.

Tasting rewards. Effects of orosensory sweet signals on human error processing.

Human research has shown interactions between rewards and cognitive control. In animal models of affective neuroscience, reward administration typically involves administering orosensory sugar signals (OSS) during caloric-deprived states. We adopted this procedure to investigate neurophysiological mechanisms of reward-cognitive control interactions in humans. We predicted that OSS would affect neurophysiological and behavioral indices of error processing oppositely, depending on the relative weight of the OSS-induced 'wanting' and 'liking' components of reward. We, therefore, conducted a double-blind, non-nutritive sweetener-controlled study with a within-subject design. Fasted (16 hr) participants (N = 61) performed a modified Flanker task to assess neurophysiological (error-related negativity [Ne/ERN]) and behavioral (post-error adaptations) measures of error processing. Non-contingent to task performance, we repeatedly administered either a sugar (glucose) or non-nutritive sweetener (aspartame) solution, which had to be expulsed after short oral stimulation to prevent post-oral effects. Consistent with our hypothesis on how 'liking' would affect Ne/ERN amplitude, we found the latter to be decreased for sugar compared to aspartame. Unexpectedly, we found post-error accuracy, instead of post-error slowing, to be reduced by sugar relative to aspartame. Our findings suggest that OSS may interact with error processing through the 'liking' component of rewards. Adopting our reward-induction procedure (i.e. administering OSS in a state of high reward sensitivity [i.e. fasting], non-contingent to task performance) might help future research investigating the neural underpinnings of reward-cognitive control interactions in humans.

Characterization of Cas12a nucleases reveals diverse PAM profiles between closely-related orthologs.

CRISPR-Cas systems comprise diverse adaptive immune systems in prokaryotes whose RNA-directed nucleases have been co-opted for various technologies. Recent efforts have focused on expanding the number of known CRISPR-Cas subtypes to identify nucleases with novel properties. However, the functional diversity of nucleases within each subtype remains poorly explored. Here, we used cell-free transcription-translation systems and human cells to characterize six Cas12a single-effector nucleases from the V-A subtype, including nucleases sharing high sequence identity. While these nucleases readily utilized each other's guide RNAs, they exhibited distinct PAM profiles and apparent targeting activities that did not track based on phylogeny. In particular, two Cas12a nucleases encoded by Prevotella ihumii (PiCas12a) and Prevotella disiens (PdCas12a) shared over 95% amino-acid identity yet recognized distinct PAM profiles, with PiCas12a but not PdCas12a accommodating multiple G's in PAM positions -2 through -4 and T in position -1. Mutational analyses transitioning PiCas12a to PdCas12a resulted in PAM profiles distinct from either nuclease, allowing more flexible editing in human cells. Cas12a nucleases therefore can exhibit widely varying properties between otherwise related orthologs, suggesting selective pressure to diversify PAM recognition and supporting expansion of the CRISPR toolbox through ortholog mining and PAM engineering.

The Effectiveness of Combat Tactical Breathing as Compared with Prolonged Exhalation.

Tactical breathing (TB) is used by military and law enforcement personnel to reduce stress and maintain psychomotor and cognitive performance in dangerous situations (Grossman and Christensen, in On combat: the psychology and physiology of deadly conflict in war and in peace, PPCT Research Publications, Belleville, 2008). So far, empirical evidence on the effectiveness of TB is limited and there are breathing techniques that are easier to learn and to apply. This study compared the effectiveness of tactical breathing and prolonged exhalation (ProlEx) under laboratory conditions. Thirty healthy participants performed a Stroop interference task under time pressure and noise distraction. Time pressure was induced with short inter-trial intervals of 350 ms and short trial durations of 1500 ms. Acoustic distraction was realised with white noise with intensity increasing from 77 to 89 dB SPL over the course of an experimental block. In a counterbalanced repeated-measures design, participants used either TB or ProlEx to reduce the induced psychological and physiological arousal. Stress reactions were assessed on the subjective level (Steyer et al., in Multidimensional mood questionnaire (MDMQ), Hogrefe, Göttingen, 1997) and on the physiological level (heart rate, heart rate variability, electrodermal activity). Results showed no significant differences between breathing techniques on the subjective level. While participants showed a lower physiological arousal in the TB condition, better performance was achieved in the ProlEx condition. Results indicate that TB may be superior in passive coping conditions, while ProlEx is more effective when active coping is required.

A facilitated diffusion mechanism establishes the Drosophila Dorsal gradient.

The transcription factor NF-κB plays an important role in the immune system, apoptosis and inflammation. Dorsal, a Drosophila homolog of NF-κB, patterns the dorsal-ventral axis in the blastoderm embryo. During this stage, Dorsal is sequestered outside the nucleus by the IκB homolog Cactus. Toll signaling on the ventral side breaks the Dorsal/Cactus complex, allowing Dorsal to enter the nucleus to regulate target genes. Fluorescent data show that Dorsal accumulates on the ventral side of the syncytial blastoderm. Here, we use modeling and experimental studies to show that this accumulation is caused by facilitated diffusion, or shuttling, of the Dorsal/Cactus complex. We also show that active Toll receptors are limiting in wild-type embryos, which is a key factor in explaining global Dorsal gradient formation. Our results suggest that shuttling is necessary for viability of embryos from mothers with compromised dorsal levels. Therefore, Cactus not only has the primary role of regulating Dorsal nuclear import, but also has a secondary role in shuttling. Given that this mechanism has been found in other, independent, systems, we suggest that it might be more prevalent than previously thought.

Anti-Tremor Action of Subtype Selective Positive Allosteric Modulators of GABAA Receptors in a Rat Model of Essential Tremors.

Essential tremor (ET) is among the most prevalent neurological disorders and the most common cause of abnormal tremors. It is characterized by postural and action tremors ranging from 4 to 12 Hz. The treatments of choice for ET are propranolol and primidone, but their use is associated with adverse effects like hypotension, depression, and cognitive impairments. Benzodiazepines, which nonselectively enhances the effect of GABA at the GABAA α1/2/3/5 receptors, have been shown to be effective in treating ET. Their use, however, is limited due to sedation, ataxia, tolerance development and memory impairment. Sedation and ataxia are attributed to the activity at the α1 subunit while cognitive impairment is ascribed to the action on the α5 subunit of the GABAA receptors. It can be hypothesized that subtype selective GABAA receptor modulators only acting via the α2, and α3 subunits may have an improved side effect profile while retaining the beneficial effects. Here, we have evaluated the effect of subtype selective GABAA α2/3/5 receptor modulators on harmaline-induced tremors in rats. The tremors were automatically quantified in tremor boxes. We show that the GABAA α2/3 subtype selective modulator NS16085 significantly and dose-dependently inhibits harmaline-induced tremors in rats, indicating that potentiation of α2- and α3-containing GABAA receptors is sufficient to ameliorate harmaline-induced tremors. These results provide the first support for a therapeutic role of a subtype selective GABAA α2/3 modulator in the treatment of ET.

Target-distractor congruency: sequential effects in a temporal flanker task.

The Congruency Sequence Effect (CSE) denotes the common finding that distractor-target Congruency Effects are reduced after incongruent compared to after congruent trials. Although the CSE is widely attributed to attentional adjustment (i.e., increasing or decreasing the bias in attentional weights regarding processing the target or distractor), unequivocal evidence for this assumption is missing. To investigate the CSE and attentional adjustment we used a temporal flanker task and intermixed a "temporal search task", in which a target stimulus occurred randomly at one of two temporal positions, corresponding to the temporal positions of the target and the distractor occurrence in the temporal flanker task. We observed a CSE that could not be explained in terms of feature sequences, distractor-related contingencies, or a strategy of reversed distractor-response priming after incongruent trials. Furthermore, following a temporal search task trial, the Congruency Effect was larger when the search target occurred on the first than on the second temporal position, demonstrating that a reduced attentional bias towards the second temporal position increased interference from a distractor presented on the first temporal position. This supports a crucial assumption of the attentional adjustment account of the CSE. Performance in the temporal search task, however, provided no evidence for attentional adjustment.

A detailed cell-free transcription-translation-based assay to decipher CRISPR protospacer-adjacent motifs.

The RNA-guided nucleases derived from the CRISPR-Cas systems in bacteria and archaea have found numerous applications in biotechnology, including genome editing, imaging, and gene regulation. However, the discovery of novel Cas nucleases has outpaced their characterization and subsequent exploitation. A key step in characterizing Cas nucleases is determining which protospacer-adjacent motif (PAM) sequences they recognize. Here, we report advances to an in vitro method based on an E. coli cell-free transcription-translation system (TXTL) to rapidly elucidate PAMs recognized by Cas nucleases. The method obviates the need for cloning Cas nucleases or gRNAs, does not require the purification of protein or RNA, and can be performed in less than a day. To advance our previously published method, we incorporated an internal GFP cleavage control to assess the extent of library cleavage as well as Sanger sequencing of the cleaved library to assess PAM depletion prior to next-generation sequencing. We also detail the methods needed to construct all relevant DNA constructs, and how to troubleshoot the assay. We finally demonstrate the technique by determining PAM sequences recognized by the Neisseria meningitidis Cas9, revealing subtle sequence requirements of this highly specific PAM. The overall method offers a rapid means to identify PAMs recognized by diverse CRISPR nucleases, with the potential to greatly accelerate our ability to characterize and harness novel CRISPR nucleases across their many uses.

The time course of distractor-based response activation with predictable and unpredictable target onset.

Electrophysiological recording in a temporal flanker task (i.e., distractors preceding the targets) has demonstrated that distractor processing is adjusted to the overall utility of the distractors. Under high utility, that is, distractors are predictive of the target/response, distractors immediately activate the corresponding response (as indicated by the lateralized readiness potential, LRP). This activation has been shown to be markedly postponed when the target predictably occurs delayed. To investigate the occurrence and time course of distractor-related response activation under conditions of unpredictable target onset, we randomly varied the stimulus-onset asynchrony (SOA) between distractors and targets and recorded the distractor-evoked LRP. When the distractor utility was high, an LRP occurred shortly after distractor presentation. In case of a long SOA the time course of this LRP was characterized by a drop back to baseline and a subsequent re-activation that reached a substantial level before target onset. These results suggest that distractor processing is characterized by sophisticated adjustments to experienced utility and temporal constraints of the task as well as by further control processes that regulate premature response activation.

Selective impairment of attentional set shifting in adults with ADHD.

BACKGROUND: Task switch protocols are frequently used in the assessment of cognitive control, both in clinical and non-clinical populations. These protocols frequently confound task switch and attentional set shift. The current study investigated the ability of adult ADHD patients to shift attentional set in the context of switching tasks. METHOD: We tested 38 adults with ADHD and 39 control adults with an extensive diagnostic battery and a task switch protocol without proactive interference. The experiment combined orthogonally task-switch vs. repetition, and attentional set shift vs. no shift. Each experimental stimulus had global and local features (Hierarchical/"Navon" stimuli), associated with corresponding attentional sets. RESULTS: ADHD patients were slower than controls in task switch trials with a simultaneous shift of attention between global/local attentional sets. This also correlated significantly with diagnostic scales for ADHD symptoms. The patients had more variable reaction times, but when the attentional set was kept constant neither were they significantly slower nor showed higher task switch costs. CONCLUSION: ADHD is associated with a deficit in flexible deployment of attention to varying sources of stimulus information.

Shifting the set of stimulus selection when switching between tasks.

Switching between tasks associated with different requirements of stimulus selection may suffer interference from persisting attentional settings or processes of reconfiguration, possibly constituting a source of task switch costs (i.e., impaired performance in task switch trials compared to task repetition trials). To explore the processes involved in task switching with and without a switch in stimulus selection requirements, we administered a task-switching paradigm that involved task-unique sets of hierarchical (Navon) stimuli, preventing stimulus-related proactive interference (i.e., impaired responding to a stimulus that was previously processed in the context of a different task), and varied, between groups of participants, whether the target stimulus level (i.e., global vs. local) was held constant or varied between the tasks. Mixing target levels impaired performance overall and increased the task switch costs. Moreover, (within-task) global-local congruency effects were larger when target levels were mixed, particularly in task switch trials, suggesting relatively stronger stimulus processing according to the attentional set of the other task. In a second phase of the experimental session, the target level was changed persistently for either one or for both tasks, presumably evoking stimulus-related proactive interference. This change yielded large task switch costs and global-local congruency effects when it resulted in mixed target levels, but not when it resulted in a constant target level. These results are consistent with the notion that stimulus-related proactive interference is reduced by constant withdrawal of attention from the perceptual dimension of the interference-eliciting stimuli.

The Distancing-Embracing model of the enjoyment of negative emotions in art reception.

Why are negative emotions so central in art reception far beyond tragedy? Revisiting classical aesthetics in the light of recent psychological research, we present a novel model to explain this much discussed (apparent) paradox. We argue that negative emotions are an important resource for the arts in general, rather than a special license for exceptional art forms only. The underlying rationale is that negative emotions have been shown to be particularly powerful in securing attention, intense emotional involvement, and high memorability, and hence is precisely what artworks strive for. Two groups of processing mechanisms are identified that conjointly adopt the particular powers of negative emotions for art's purposes. The first group consists of psychological distancing mechanisms that are activated along with the cognitive schemata of art, representation, and fiction. These schemata imply personal safety and control over continuing or discontinuing exposure to artworks, thereby preventing negative emotions from becoming outright incompatible with expectations of enjoyment. This distancing sets the stage for a second group of processing components that allow art recipients to positively embrace the experiencing of negative emotions, thereby rendering art reception more intense, more interesting, more emotionally moving, more profound, and occasionally even more beautiful. These components include compositional interplays of positive and negative emotions, the effects of aesthetic virtues of using the media of (re)presentation (musical sound, words/language, color, shapes) on emotion perception, and meaning-making efforts. Moreover, our Distancing-Embracing model proposes that concomitant mixed emotions often help integrate negative emotions into altogether pleasurable trajectories.

Negative emotions in art reception: Refining theoretical assumptions and adding variables to the Distancing-Embracing model.

While covering all commentaries, our response specifically focuses on the following issues: How can the hypothesis of emotional distancing (qua art framing) be compatible with stipulating high levels of felt negative emotions in art reception? Which concept of altogether pleasurable mixed emotions does our model involve? Can mechanisms of predictive coding, social sharing, and immersion enhance the power of our model?

Utility-based early modulation of processing distracting stimulus information.

Humans are selective information processors who efficiently prevent goal-inappropriate stimulus information to gain control over their actions. Nonetheless, stimuli, which are both unnecessary for solving a current task and liable to cue an incorrect response (i.e., "distractors"), frequently modulate task performance, even when consistently paired with a physical feature that makes them easily discernible from target stimuli. Current models of cognitive control assume adjustment of the processing of distractor information based on the overall distractor utility (e.g., predictive value regarding the appropriate response, likelihood to elicit conflict with target processing). Although studies on distractor interference have supported the notion of utility-based processing adjustment, previous evidence is inconclusive regarding the specificity of this adjustment for distractor information and the stage(s) of processing affected. To assess the processing of distractors during sensory-perceptual phases we applied EEG recording in a stimulus identification task, involving successive distractor-target presentation, and manipulated the overall distractor utility. Behavioral measures replicated previously found utility modulations of distractor interference. Crucially, distractor-evoked visual potentials (i.e., posterior N1) were more pronounced in high-utility than low-utility conditions. This effect generalized to distractors unrelated to the utility manipulation, providing evidence for item-unspecific adjustment of early distractor processing to the experienced utility of distractor information.

Bad beauty: Aesthetic judgments are influenced by references to morally contentious content in photographs.

Affective responses can influence evaluative judgments, but how are subjective beauty ratings affected by references to morally contentious elements in aesthetic stimuli? In an online experiment (N = 460), we investigated the relationship between two types of descriptive texts (Neutral vs. Negative) and the beauty ratings of 25 photographs that depict sources of environmental pollution. For each photograph, the neutral descriptive text contained general information, whereas the negative descriptive text addressed the pollution source. Further, we explored whether this relationship is mediated by changes in positive and negative affect, and how it interacts with the biospheric values of participants. Our results showed that (1) participants in the Negative Condition rated the photographs as less beautiful than in the Neutral Condition, (2) this relationship was partially mediated by changes in negative affect, and (3) in the Negative Condition, participants with higher levels of biospheric values rated the photographs as less beautiful. Our results indicate that individual values, as well as affective responses induced by aesthetic stimuli, directly influence subjective beauty. This aligns with current theoretical frameworks and fills a gap in experimental research. Finally, we discuss limitations and directions for future studies. PSYCHINFO CLASSIFICATION CODE: 2340.

Assessing the desire for aesthetics: Adaptation and validation of the desire for aesthetics scale in German (DFAS-G).

OBJECTIVES: Individuals vary in their levels of Desire for Aesthetics, ranging from superficial interest to profound immersion. However, there is currently a lack of measurement tools available to assess Desire for Aesthetics across various domains within German-speaking populations. Addressing this gap, the present study aimed to develop the Desire for Aesthetics Scale in German (DFAS-G) and evaluate its psychometric properties to comprehensively assess aesthetic preferences and inclinations. METHODS: We conducted three consecutive studies and collected three samples of students and university employees. In Study 1 (N = 317), we selected a translated item pool using the forward-backward translation method, conducted cultural adaptation of the items, and assessed the scale's factor structure through an exploratory factor analysis. In Study 2 (N = 304), we revised and cross-validated the scale using confirmatory factor analysis and examined the final 37-item DFAS-G for reliability and validity. In Study 3 (N = 300), we further examined the scales construct validity and assessed retest reliability 291 days after initial testing. RESULTS: The factor analysis conducted in Study 1 resulted in a four-factor solution representing the Desire for Aesthetics in four domains: Visual Arts, Individuals, Music, and Habitat. We found good internal consistency for the global scale (Cronbach's alpha = 0.87) and all subscales (0.74 to 0.82). We also found construct validity with Art Interest and facets of the Big Five personality trait Openness and provided first evidence of criterion validity and incremental validity of the DFAS-G global score toward Aesthetic Perception and Visual Arts toward Art Interest in Study 2. We further found evidence of convergent validity with the Openness facet Aesthetics, discriminant validity with the facet Values, and high test-retest reliability (0.85 to 1.00) in Study 3. CONCLUSIONS: The present article provides a scale for measuring DfA in German-speaking populations and first steps of its validation. The DFAS-G extends the repertoire of options for psychological research on individual differences in aesthetics appreciation. Follow-up studies could provide further evidence of the subscale's validity and equivalence to the original scale.

The impact of crossmodal predictions on the neural processing of aesthetic stimuli.

Neuroaesthetic research has focused on neural predictive processes involved in the encounter with art stimuli or the related evaluative judgements, and it has been mainly conducted unimodally. Here, with electroencephalography, magnetoencephalography and an affective priming protocol, we investigated whether and how the neural responses to non-representational aesthetic stimuli are top-down modulated by affective representational (i.e. semantically meaningful) predictions between audition and vision. Also, the neural chronometry of affect processing of these aesthetic stimuli was investigated. We hypothesized that the early affective components of crossmodal aesthetic responses are dependent on the affective and representational predictions formed in another sensory modality resulting in differentiated brain responses, and that audition and vision indicate different processing latencies for affect. The target stimuli were aesthetic visual patterns and musical chords, and they were preceded by a prime from the opposing sensory modality. We found that early auditory-cortex responses to chords were more affected by valence than the corresponding visual-cortex ones. Furthermore, the assessments of visual targets were more facilitated by affective congruency of crossmodal primes than the acoustic targets. These results indicate, first, that the brain uses early affective information for predictively guiding aesthetic responses; second, that an affective transfer of information takes place crossmodally, mainly from audition to vision, impacting the aesthetic assessment. This article is part of the theme issue 'Art, aesthetics and predictive processing: theoretical and empirical perspectives'.

Preparation and persistence of deploying attention to locations or stimulus structures: Evidence from intermixed probe trials.

Attention can be directed to the global or local level of a visual stimulus (i.e., Navon figure). Previous studies yielded reliable trial-to-trial level switch costs (i.e., worse performance when responding to the other level than on a previous trial), even though level cueing effects indicated anticipatory deployment of attention to the upcoming target level. To investigate the interplay of attentional preparation and persistence, we applied a probe trial method assumed to ensure a high degree of preparation for the upcoming target level and minimizing stimulus-specific proactive interference. Mirroring previous findings obtained in the domain of spatial attention, we found evidence for anticipatory attentional focusing on global/local target levels but not for persistence of the attentional set adopted on the previous trial. In a second experiment, we prevented preparation for upcoming attentional demands (in both global-local and spatial attention tasks). This resulted in the modulation of performance (in critical probe trials) by the attentional demands of the predecessor trial. Together, our findings demonstrate sensitivity of the probe trial method for attentional persistence and raise the possibility that such persistence can be completely eliminated by sufficiently strong preparation for the attentional demands of the following trial.

Flexible processing of distractor stimuli under stress.

Acute stress is assumed to affect executive processing of stimulus information, although extant studies have yielded heterogeneous findings. The temporal flanker task, in which a target stimulus is preceded by a distractor of varying utility, offers a means of investigating various components involved in the adjustment of information processing and conflict control. Both behavioral and EEG data obtained with this task suggest stronger distractor-related response activation in conditions associated with higher predictivity of the distractor for the upcoming target. In two experiments we investigated distractor-related processing and conflict control after inducing acute stress (Trier Social Stress Test). Although the stressed groups did not differ significantly from unstressed control groups concerning behavioral markers of attentional adjustment (i.e., Proportion Congruent Effect), or event-related sensory components in the EEG (i.e., posterior P1 and N1), the lateralized readiness potential demonstrated reduced activation evoked by (predictive) distractor information under stress. Our results suggest flexible adjustment of attention under stress but hint at decreased usage of nominally irrelevant stimulus information for biasing response selection.