Toddlers' affective responses to sociomoral scenes: Insights from physiological measures.

A growing literature suggests that preverbal infants are sensitive to sociomoral scenes and prefer prosocial agents over antisocial agents. It remains unclear, however, whether and how emotional processes are implicated in infants' responses to prosocial/antisocial actions. Although a recent study found that infants and toddlers showed more positive facial expressions after viewing helping (vs. hindering) events, these findings were based on naïve coder ratings of facial activity; furthermore, effect sizes were small. The current studies examined 18- and 24-month-old toddlers' real-time reactivity to helping and hindering interactions using three physiological measures of emotion-related processes. At 18 months, activity in facial musculature involved in smiling/frowning was explored via facial electromyography (EMG). At 24 months, stress (sweat) was explored via electrodermal activity (EDA). At both ages, arousal was explored via pupillometry. Behaviorally, infants showed no preferences for the helper over the hinderer across age groups. EMG analyses revealed that 18-month-olds showed higher corrugator activity (more frowning) during hindering (vs. helping) actions, followed by lower corrugator activity (less frowning) after hindering (vs. helping) actions finished. These findings suggest that antisocial actions elicited negativity, perhaps followed by brief disengagement. EDA analyses revealed no significant event-related differences. Pupillometry analyses revealed that both 18- and 24-month-olds' pupils were smaller after viewing hindering (vs. helping), replicating recent evidence with 5-month-olds and suggesting that toddlers also show less arousal following hindering than following helping. Together, these results provide new evidence with respect to whether and how arousal/affective processes are involved when infants process sociomoral scenarios.

The power of the written word: Comparing word meaning inferencing from auditory and written modalities throughout grade school.

Previous research indicates that word learning from auditory contexts may be more effective than written context at least through fourth grade. However, no study has examined contextual differences in word learning in older school-aged children when reading abilities are more developed. Here we examined developmental differences in children's ability to deduce the meanings of unknown words from the surrounding linguistic context in the auditory and written modalities and sought to identify the most important predictors of success in each modality. A total of 89 children aged 8-15 years were randomly assigned to either read or listen to a narrative that included eight novel words, with five exposures to each novel word. They then completed three posttests to assess word meaning inferencing. Children across all ages performed better in the written modality. Vocabulary was the only significant predictor of success on the word inferencing task. Results indicate support for written stimuli as the most effective modality for novel word meaning deduction. Our findings suggest that the presence of orthographic information facilitates novel word learning even for early, less proficient readers.

Intuitive mapping between nonsymbolic quantity and observed action across development.

Adults' concurrent processing of numerical and action information yields bidirectional interference effects consistent with a cognitive link between these two systems of representation. This link is in place early in life: infants create expectations of congruency across numerical and action-related stimuli (i.e., a small [large] hand aperture associated with a smaller [larger] numerosity). Although these studies point to a developmental continuity of this mapping, little is known about the later development and thus how experience shapes such relationships. We explored how number-action intuitions develop across early and later childhood using the same methodology as in adults. We asked 3-, 6-, and 8-year-old children, as well as adults, to relate the magnitude of an observed action (a static hand shape, open vs. closed, in Experiment 1; a dynamic hand movement, opening vs. closing, in Experiment 2) to either a small or large nonsymbolic quantity (numerosity in Experiment 1 and numerosity and/or object size in Experiment 2). From 6 years of age, children started performing in a systematic congruent way in some conditions, but only 8-year-olds (added in Experiment 2) and adults performed reliably above chance in this task. We provide initial evidence that early intuitions guiding infants' mapping between magnitude across nonsymbolic number and observed action are used in an explicit way only from late childhood, with a mapping between action and size possibly being the most intuitive. An initial coarse mapping between number and action is likely modulated with extensive experience with grasping and related actions directed to both arrays and individual objects.

Truthful yet misleading: Elementary second-order deception in school-age children and its sociocognitive correlates.

In highly competitive contexts, deceptive intentions might be transparent, so conveying only false information to the opponent can become a predictable strategy. In such situations, alternating between truths and lies (second-order lying behavior) represents a less foreseeable option. The current study investigated the development of 8- to 10-year-old children's elementary second-order deception in relation to their attribution of ignorance (first- and second-order ignorance) and executive functions (inhibitory control, shifting ability, and verbal working memory). An adapted version of the hide-and-seek paradigm was used to assess children's second-order lie-telling, in which children were asked to hide a coin in either of their hands. Unlike the standard paradigm, the opponent did not consistently look for the coin in the location indicated by the children, so children needed to switch between telling simple lies and truths (elementary second-order lies about the coin location) to successfully deceive the recipient. The results showed that older children were less likely to tell elementary second-order lies. However, across the sample, when children decided to lie, this ability was positively related to their second-order ignorance attribution and their verbal working memory. Moreover, we obtained preliminary evidence for the presence of a habituation effect in second-order lying, with children being more accurate and having less variability in their truthful-to-deceive responses (this being the more frequently elicited response) than when telling lies to deceive. Our findings could have implications for understanding the mechanisms underlying children's ability to alternate between truths and lies to deceive.

Holding multiple category representations: The role of age, theory of mind, and rule switching in children's developing cross-classification abilities.

Cross-classification, the ability to categorize multifaceted entities in many ways, is a remarkable cognitive milestone for children. Past work has focused primarily on documenting the timeline for when children reach cross-classification competence. However, it is not well understood what cognitive factors underpin children's improvements. The current study aimed to examine the contributions of age, theory of mind, and rule switching to children's cross-classification development. We tested 3- to 5-year-old children (N = 75) using a cross-classification task, the Theory of Mind Task Battery, and the Three-Dimensional Change Card Sort test. The results revealed that age and theory of mind predict children's cross-classification over and above the effects of rule switching. The results also revealed that advanced-level theory of mind reasoning is a particularly strong predictor of cross-classification development. These findings increase understanding of cross-classification within children's broader cognitive development.

No influence of regular rhythmic priming on grammaticality judgment and sentence comprehension in English-speaking children.

A growing body of research has demonstrated the association between music and language, particularly between rhythm and grammar skills in children. A compelling piece of evidence for the influence of music on language comes from findings that a brief exposure to regular musical rhythm improved subsequent syntactic language performance in children. Nevertheless, those observations were made on one particular task, i.e., grammaticality judgment, mostly with French-speaking children. Here, we sought to corroborate and extend the rhythmic priming effect with English-speaking children aged 7 to 12 years who underwent two different syntactic tasks on spoken sentences: one involving judgment on morphosyntactic well-formedness (grammaticality judgment) and the other requiring noun-verb relation analysis (sentence comprehension), both following either regular or irregular rhythmic priming. Half of the children were administered synthetic speech stimuli (Experiment 1), and the other half were presented with natural speech (Experiment 2). Across the two experiments, we did not find any rhythmic priming effect; children's performance on both the grammaticality judgment and sentence comprehension tasks was comparable irrespective of the regularity in prior rhythms. These results imply that the positive influence of regular rhythmic priming on syntactic processing may be confined to specific language or age populations, warranting further investigation.

Motor constraints on infant search: The impact of reaching around a barrier on search performance.

This study investigated the impact of perceptual-motor context on a classic paradigm used to assess cognitive-spatial reasoning. Specifically, this project explored the effect on search behavior of reaching around a barrier versus not reaching around a barrier during the A portion in the B phase of the well-known A-not-B task. In examining 8- and 16-month-old infants, this study found that both age groups demonstrated poorer performance on A trials when needing to reach around a barrier than when there was no barrier present. More interestingly, for the younger infants, needing to reach around a barrier on A trials led to better performance on B trials relative to infants who did not reach around a barrier. Older infants, however, showed no difference in B trial performance. These results demonstrate that inducing constraints on reaching (e.g., reaching around a barrier) has a significant impact on search behavior, a finding that theoretically fits with a literature demonstrating a fundamental role in behavior of perceptual and motor influences in A-not-B behavior.

An experimental task to measure preschool children's frustration induced by having to wait unexpectedly: The role of sensitivity to delay and culture.

The ability to manage frustration induced by having to wait for valued outcomes emerges across childhood and is an important marker of self-regulatory capacity. However, approaches to measure this capacity in preschool children are lacking. In this study, we introduced a new task, the Preschool Delay Frustration Task (P-DeFT), designed specifically to identify children's behavioral and emotional markers of waiting-induced frustration during the imposed wait period and after the release from waiting. We then explored how waiting-induced frustration relates to individual differences in delay sensitivity and whether it differs between two cultural groups thought to have different attitudes toward children's conduct and performance: Hong Kong (HK) and the United Kingdom (UK). A total of 112 preschool children (mean age = 46.22 months) completed the P-DeFT in a quiet laboratory. Each trial had two stages; first, a button press elicited a Go signal; second, this Go signal allowed children to go to a "supermarket" to pick a target toy. On most trials, the Go signal occurred immediately on the first press. On 6 trials, an unexpected/unsignaled 5- or 10-s pre-Go-signal period was imposed. Frustration was indexed by performance (button presses and press duration), behavioral agitation, and negative affect during the pre-Go-signal wait period and the post-Go-signal shopping task. Parents rated their children's delay sensitivity. Waiting-related frustration expressed during both the pre-Go-signal wait period and the post-Go-signal task varied with (a) the length of wait and (b) individual differences in parent-rated delay sensitivity. UK children displayed more negative affect during delay than their HK counterparts, although the relationship between delay sensitivity and frustration was culturally invariant.

Auditory attention influences trajectories of symbol-speech sound learning in children with and without dyslexia.

The acquisition of letter-speech sound correspondences is a fundamental process underlying reading development, one that could be influenced by several linguistic and domain-general cognitive factors. In the current study, we mimicked the first steps of this process by examining behavioral trajectories of audiovisual associative learning in 110 7- to 12-year-old children with and without dyslexia. Children were asked to learn the associations between eight novel symbols and native speech sounds in a brief training and subsequently read words and pseudowords written in the artificial orthography. We then investigated the influence of auditory attention as one of the putative domain-general factors influencing associative learning. To this aim, we assessed children with experimental measures of auditory sustained selective attention and interference control. Our results showed shallower learning trajectories in children with dyslexia, especially during the later phases of the training blocks. Despite this, children with dyslexia performed similarly to typical readers on the post-training reading tests using the artificial orthography. Better auditory sustained selective attention and interference control skills predicted greater response accuracy during training. Sustained selective attention was also associated with the ability to apply these novel correspondences in the reading tests. Although this result has the limitations of a correlational design, it denotes that poor attentional skills may constitute a risk during the early stages of reading acquisition, when children start to learn letter-speech sound associations. Importantly, our findings underscore the importance of examining dynamics of learning in reading acquisition as well as individual differences in more domain-general attentional factors.

Children are eager to take credit for prosocial acts, and cost affects this tendency.

We report two experiments on children's tendency to enhance their reputations through communicative acts. In the experiments, 4-year-olds (N = 120) had the opportunity to inform a social partner that they had helped him in his absence. In a first experiment, we pitted a prosocial act ("Let's help clean up for Doggie!") against an instrumental act ("Let's move these out of our way"). Children in the prosocial condition were quicker to inform their partner of the act and more likely to protest when another individual was given credit for it. In a second experiment, we replicated the prosocial condition but with a new manipulation: high-cost versus low-cost helping. We manipulated both the language surrounding cost (i.e., "This will be pretty tough to clean up" vs. "It will be really easy to clean this up") and how difficult the task itself was. As predicted, children in the high-cost condition were quicker to inform their partner of the act and more likely to take back credit for it. These results suggest that even 4-year-old children make active attempts to elicit positive reputational judgments for their prosocial acts, with cost as a moderating factor.

Children's use of reasoning by exclusion to infer objects' identities in working memory.

Reasoning by exclusion allows us to form more complete representations of our environments, "filling in" inaccessible information by ruling out known alternatives. In two experiments (Experiment 1: N = 34 4- to 6-year-olds; Experiment 2: N = 85 4- to 8-year-olds), we examined children's ability to use reasoning by exclusion to infer the identity of an unknown object and investigated the role of working memory in this ability. Children were asked to encode a set of objects that were then hidden, and after a brief retention interval children were asked to select the identity of the object hidden in one of the locations from two alternatives. On some trials, all the images were visible during encoding, so selecting the correct identity when probed required successful working memory storage and retrieval. On other trials, all but one of the images was visible during encoding, so selecting the correct identity when probed also required maintaining a representation of an unknown object in working memory and then using reasoning by exclusion to fill in the missing information retroactively to complete that representation by ruling out known alternatives. To investigate the working memory cost of exclusive reasoning, we manipulated the working memory demands of the task. Our results suggest that children can use reasoning by exclusion to retroactively assign an identity to an incomplete object representation at least by 4 years of age but that this ability incurs some cognitive cost, which eases with development. These results provide new insights into children's representational capacities and on the foundational building blocks of fully developed exclusive reasoning.

Active help-seeking and metacognition interact in supporting children's retention of science facts.

Determining when to ask for help is a critical self-regulated strategy that can benefit children's learning. Despite its importance, we have a limited understanding about the developmental mechanisms that support adaptive help-seeking. In the current preregistered study, predominately White children aged 8 to 13 years (N = 69, ngirls = 37) had the option to seek help during an online science learning task. Results revealed that children's ability to adaptively seek help improved throughout childhood and early adolescence. Critically, developing metacognitive skills contributed to greater help-related memory benefits (compared with conditions where help was not previously available). Overall, these findings highlight the role of metacognition in children's ability to adaptively seek and benefit from help during online science learning.

Functional In Vivo Imaging of Macrophages.

Resident tissue macrophages (RTMs) are specialized phagocytes that are widely distributed throughout the body and are responsible for maintaining homeostasis. Recent advances in experimental techniques have enabled us to gain a greater insight into the actual in vivo biology of RTMs by observing their spatiotemporal dynamics directly in their native environment. Here, we detail a method for live tracking macrophages in a prototypical stromal tissue with high spatial and temporal resolution and great experimental versatility. Our approach builds on a custom intravital imaging platform and straightforward surgical preparation to gain access to an intact stromal compartment in order to analyze the morphological and behavioral dynamics of RTMs at single-cell resolution before and after experimental intervention. Furthermore, our versatile approach can also be utilized for live visualization of intracellular signaling and even for tracking cell organelles at subcellular resolution, and can be combined with downstream analyses such as multiplex confocal imaging, providing a unique insight into macrophage biology in vivo.

Monitoring of Inflammasome Activation of Macrophages and Microglia In Vitro, Part 1: Cell Preparation and Inflammasome Stimulation.

Inflammasomes are intracellular, multiprotein supercomplexes that mediate a post-translational inflammatory response to both pathogen and endogenous danger signals. They consist of a sensor, the adapter ASC, and the protease caspase 1 and, following their activation, lead to cl1ß, as well as lytic cell death. Due to this potent inflammatory capacity, understanding inflammasome biology is important in many pathological conditions. It is increasingly clear that inflammasomes are particularly relevant in macrophages, which express a diverse range of inflammasome sensors. In these two chapters, we detail methods to isolate and differentiate human macrophages, murine bone marrow-derived macrophages, and murine microglia and stimulate the inflammasomes known to be expressed in macrophages, including the AIM2, NLRP3, NLRC4, NLRP1, and non-canonical inflammasomes. Furthermore, we describe the methodology required to measure the various results of inflammasome activation including ASC speck formation, monitoring lytic cell death and cytokine release, as well as caspase-1 activation.

Adaptation of Human iPSC-Derived Macrophages Toward an Alveolar Macrophage-Like Phenotype Post-Intra-Pulmonary Transfer into Murine Models.

Alveolar macrophages (AMs) represent crucial immune cells in the bronchioalveolar space of the lung. Given the important role in the host defense machinery and lung tissue homeostasis, AMs have been linked to a variety of diseases and thus represent a promising target cell type for novel therapies. The emerging importance of AM underlines the necessity to isolate and/or generate proper cellular models, which facilitate basic biology and translational science. As of yet, most studies focus on the derivation of AM from the murine system. This chapter introduces the use of human-induced pluripotent stem cell (iPSC)-derived primitive macrophages, which can be further matured towards an AM-like phenotype upon intra-pulmonary transfer into mice. We will give a brief overview on the generation of primitive iPSC-derived macrophages, which is followed by a detailed, step-by-step description of the intra-pulmonary transfer of cells and the follow-up procedures needed to isolate the iPSC-derived, AM-like cells from the lungs post-transfer. The chapter provides an alternative approach to derive human AM-like cells, which can be used to study human AM biology and to investigate novel therapeutic interventions using primitive macrophages from iPSC.

Functional Genomics of Novel Rhabdomyosarcoma Fusion-Oncogenes Using Zebrafish.

Clinical sequencing efforts continue to identify novel putative oncogenes with limited strategies to perform functional validation in vivo and study their role in tumorigenesis. Here, we present a pipeline for fusion-driven rhabdomyosarcoma (RMS) in vivo modeling using transgenic zebrafish systems. This strategy originates with novel fusion-oncogenes identified from patient samples that require functional validation in vertebrate systems, integrating these genes into the zebrafish genome, and then characterizing that they indeed drive rhabdomyosarcoma tumor formation. In this scenario, the human form of the fusion-oncogene is inserted into the zebrafish genome to understand if it is an oncogene, and if so, the underlying mechanisms of tumorigenesis. This approach has been successful in our models of infantile rhabdomyosarcoma and alveolar rhabdomyosarcoma, both driven by respective fusion-oncogenes, VGLL2-NCOA2 and PAX3-FOXO1. Our described zebrafish platform is a rapid method to understand the impact of fusion-oncogene activity, divergent and shared fusion-oncogene biology, and whether any analyzed pathways converge for potential clinically actionable targets.

Delivering Traumatic Brain Injury to Larval Zebrafish.

We describe a straightforward, scalable method for administering traumatic brain injury (TBI) to zebrafish larvae. The pathological outcomes appear generalizable for all TBI types, but perhaps most closely model closed-skull, diffuse lesion (blast injury) neurotrauma. The injury is delivered by dropping a weight onto the plunger of a fluid-filled syringe containing zebrafish larvae. This model is easy to implement, cost-effective, and provides a high-throughput system that induces brain injury in many larvae at once. Unique to vertebrate TBI models, this method can be used to deliver TBI without anesthetic or other metabolic agents. The methods simulate the main aspects of traumatic brain injury in humans, providing a preclinical model to study the consequences of this prevalent injury type and a way to explore early interventions that may ameliorate subsequent neurodegeneration. We also describe a convenient method for executing pressure measurements to calibrate and validate this method. When used in concert with the genetic tools readily available in zebrafish, this model of traumatic brain injury offers opportunities to examine many mechanisms and outcomes induced by traumatic brain injury. For example, genetically encoded fluorescent reporters have been implemented with this system to measure protein misfolding and neural activity via optogenetics.

Methods to Study Liver Disease Using Zebrafish Larvae.

Liver disease affects millions of people worldwide, and the high morbidity and mortality is attributed in part to the paucity of treatment options. In many cases, liver injury self-resolves due to the remarkable regenerative capacity of the liver, but in cases when regeneration cannot compensate for the injury, inflammation and fibrosis occur, creating a setting for the emergence of liver cancer. Whole animal models are crucial for deciphering the basic biological underpinnings of liver biology and pathology and, importantly, for developing and testing new treatments for liver disease before it progresses to a terminal state. The cellular components and functions of the zebrafish liver are highly similar to mammals, and zebrafish develop many diseases that are observed in humans, including toxicant-induced liver injury, fatty liver, fibrosis, and cancer. Therefore, the widespread use of zebrafish larvae for studying the mechanisms of these pathologies and for developing potential treatments necessitates the optimization of experimental approaches to assess liver disease in this model. Here, we describe protocols using staining methods, imaging, and gene expression analysis to assess liver injury, fibrosis, and preneoplastic changes in the liver of larval zebrafish.

Antifouling modification for high-performance isolation of circulating tumor cells.

Circulating tumor cells (CTCs), which shed from solid tumor tissue into blood circulatory system, have attracted wide attention as a biomarker in the early diagnosis and prognosis of cancer. Given their potential significance in clinics, many platforms have been developed to separate CTCs. However, the high-performance isolation of CTCs remains significant challenges including achieving the sensitivity and specificity necessary due to their extreme rarity and severe biofouling in blood, such as billions of background cells and various proteins. With the advancement of CTCs detection technologies in recent years, the highly efficient and highly specific detection platforms for CTCs have gradually been developed, resulting in improving CTC capture efficiency, purity and sensitivity. In this review, we systematically describe the current strategies with surface modifications by utilizing the antifouling property of polymer, peptide, protein and cell membrane for high-performance enrichment of CTCs. To wrap up, we discuss the substantial challenges facing by current technologies and the potential directions for future research and development.

High-sensitive and rapid electrochemical detection of miRNA-31 in saliva using Cas12a-based 3D nano-harvester with improved trans-cleavage efficiency.

Salivary miRNA-31 is a reliable diagnostic marker for early-stage oral squamous cell carcinoma (OSCC), but accurate detection of miRNA-31 in saliva samples is a challenge because of its low level and high sequence homology. The CRISPR/Cas12a system has the exceptional potential to enable simple nucleic acid analysis but suffers from low speed and sensitivity. To achieve rapid and high-sensitive detection of miRNA-31 using the CRISPR/Cas12a system, a Cas12a-based nano-harvester activated by a polymerase-driven DNA walker, named as dual 3D nanorobots, was developed. The target walked rapidly on the surface of DNA hairpin-modified magnetic nanoparticles driven by DNA polymerase, generating numerous double-strand DNA (dsDNA). Then, the Cas12a bound to the generated dsDNA for activating its trans-cleavage activity, forming 3D nano-harvester. Subsequently, the harvester cut and released methylene blue-labeled DNA hairpins immobilized on the sensing interface, leading to the change in electrochemical signal. We found that the trans-cleavage activity of the harvester was higher than the conventional CRISPR/Cas12a system. The developed dual 3D nanorobots could enable rapid (detection time within 60 min), high-sensitive (detection limit of femtomolar), and specific analysis of miRNA-31 in saliva samples. Thus, our established electrochemical biosensing strategy has great potential for early diagnosis of OSCC.