Suggested role for neutrophil extracellular trap formation in Ewing sarcoma immune microenvironment.

Ewing sarcoma (EWS) is a highly aggressive cancer with a survival rate of 70%-80% for patients with localized disease and under 30% for those with metastatic disease. Tumor-infiltrating neutrophils (TIN) can generate extracellular net-like DNA structures known as neutrophil extracellular traps (NETs). However, little is known about the presence and prognostic significance of tumor-infiltrating NETs in EWS. Herein, we investigated 46 patients diagnosed with EWS and treated in the Tel Aviv Medical Center between 2010 and 2021. TINs and NETs were identified in diagnostic biopsies of EWS by immunofluorescence. In addition, NETs were investigated in neutrophils isolated from peripheral blood samples of EWS patients at diagnosis and following neoadjuvant chemotherapy. The relationships between the presence of TINs and NETs, pathological and clinical features, and outcomes were analyzed. Our results demonstrate that TIN and NETs at diagnosis were higher in EWS patients with metastatic disease compared with those with local disease. High NET formation at diagnosis predicted poor response to neoadjuvant chemotherapy, relapse, and death from disease (p < 0.05). NET formation in peripheral blood samples at diagnosis was significantly elevated among patients with EWS compared with pediatric controls and decreased significantly following neoadjuvant chemotherapy. In conclusion, NET formation seems to have a role in the EWS immune microenvironment. Their presence can refine risk stratification, predict chemotherapy resistance and survival, and serve as a therapeutic target in patients with EWS.

Language experience matters for the emergence of early numerical concepts.

Research has shown a link between the acquisition of numerical concepts and language, but exactly how linguistic input matters for numerical development remains unclear. Here, we examine both symbolic (number word knowledge) and non-symbolic (numerical discrimination) numerical abilities in a population in which access to language is limited early in development-oral deaf and hard of hearing (DHH) preschoolers born to hearing parents who do not know a sign language. The oral DHH children demonstrated lower numerical discrimination skills, verbal number knowledge, conceptual understanding of the word "more", and vocabulary relative to their hearing peers. Importantly, however, analyses revealed that group differences in the numerical tasks, but not vocabulary, disappeared when differences in the amount of time children had had auditory access to spoken language input via hearing technology were taken into account. Results offer insights regarding the role language plays in emerging number concepts.

Neutrophils extracellular traps formation may serve as a biomarker for disease activity in oligoarticular juvenile idiopathic arthritis: a pilot study.

BACKGROUND: Juvenile idiopathic arthritis (JIA) is the most common chronic rheumatic disease in children, causing significant morbidity. Despite the dramatic improvement in treatment, many patients do not achieve complete remission, and biomarkers for subclinical disease, flares, and response to treatment are lacking. Neutrophils and neutrophil extracellular traps (NETs) play key roles in the pathogenesis of autoimmune and inflammatory conditions. In this study, we characterized neutrophil enzyme activity and NETs formation in oligoarticular and polyarticular JIA and explored their association with disease activity. METHODS: Neutrophils from 6 healthy controls and 7 patients with oligoarticular and polyarticular JIA were freshly isolated at time of diagnosis and after glucocorticoid intra-articular injection. Enzymatic activity of neutrophil granular enzymes was monitored by colorimetry and PMA-activated NETs formation was assessed using fluorescent microscopy. RESULTS: In this pilot and feasibility study, we revealed that NETs were significantly increased in oligoarticular JIA patients at time of diagnosis compared to healthy controls. Anti-inflammatory treatment using intra-articular steroid injection normalized NETs formation in these patients. Correlation between NETs formation and clinical Juvenile Activity Disease Activity Score-10 (cJADAS-10) was linear and significant (P = 0.007) in oligo but not in poly JIA patients. CONCLUSIONS: This is the first study exploring the link of NETs formation with oligo and poly JIA activity. We demonstrated a statistically significant linear correlation between cJADAS-10 and NETs formation in oligo but not in poly JIA patients. Hence, we suggest that NETs may reflect clinical disease activity in JIA, and may serve as a putative biomarker. Further work is needed to validate these initial results and determine the dynamics of NETs formation in JIA.

The development of early numeracy in deaf and hard of hearing children acquiring spoken language.

Most deaf and hard-of-hearing (DHH) children are born to hearing parents and steered toward spoken rather than signed language, introducing a delay in language access. This study investigated the effects of this delay on number acquisition. DHH children (N = 44, meanage  = 58 months, 21F, >50% White) and typically-hearing (TH) children (N = 79, meanage  = 49 months, 51F, >50% White) were assessed on number and language in 2011-13. DHH children showed similar trajectories to TH children but delayed timing; a binary logistic regression showed that the odds of being a cardinal-principle (CP) knower were 17 times higher for TH children than DHH children, controlling for age (d = .69). Language fully mediated the association between deaf/hearing group and number knowledge, suggesting that language access sets the pace for number acquisition.

Acquisition of the counting principles during the subset-knower stages: Insights from children's errors.

Studies on children's understanding of counting examine when and how children acquire the cardinal principle: the idea that the last word in a counted set reflects the cardinal value of the set. Using Wynn's (1990) Give-N Task, researchers classify children who can count to generate large sets as having acquired the cardinal principle (cardinal-principle-knowers) and those who cannot as lacking knowledge of it (subset-knowers). However, recent studies have provided a more nuanced view of number word acquisition. Here, we explore this view by examining the developmental progression of the counting principles with an aim to elucidate the gradual elements that lead to children successfully generating sets and being classified as CP-knowers on the Give-N Task. Specifically, we test the claim that subset-knowers lack cardinal principle knowledge by separating children's understanding of the cardinal principle from their ability to apply and implement counting procedures. We also ask when knowledge of Gelman & Gallistel's (1978) other how-to-count principles emerge in development. We analyzed how often children violated the three how-to-count principles in a secondary analysis of Give-N data (N = 86). We found that children already have knowledge of the cardinal principle prior to becoming CP-knowers, and that understanding of the stable-order and word-object correspondence principles likely emerged earlier. These results suggest that gradual development may best characterize children's acquisition of the counting principles and that learning to coordinate all three principles represents an additional step beyond learning them individually.

Numerical cognition needs more and better distinctions, not fewer.

We agree that the approximate number system (ANS) truly represents number. We endorse the authors' conclusions on the arguments from confounds, congruency, and imprecision, although we disagree with many claims along the way. Here, we discuss some complications with the meanings that undergird theories in numerical cognition, and with the language we use to communicate those theories.

Split Chloramphenicol Acetyl-Transferase Assay Reveals Self-Ubiquitylation-Dependent Regulation of UBE3B.

Split reporter protein-based genetic section systems are widely used to identify and characterize protein-protein interactions (PPI). The assembly of split markers that antagonize toxins, rather than required for synthesis of missing metabolites, facilitates the seeding of high density of cells and selective growth. Here we present a newly developed split chloramphenicol acetyltransferase (split-CAT) -based genetic selection system. The N terminus fragment of CAT is fused downstream of the protein of interest and the C terminus fragment is tethered upstream to its postulated partner. We demonstrate the system's advantages for the study of PPIs. Moreover, we show that co-expression of a functional ubiquitylation cascade where the target and ubiquitin are tethered to the split-CAT fragments results in ubiquitylation-dependent selective growth. Since proteins do not have to be purified from the bacteria and due to the high sensitivity of the split-CAT reporter, detection of challenging protein cascades and post-translation modifications is enabled. In addition, we demonstrate that the split-CAT system responds to small molecule inhibitors and molecular glues (GLUTACs). The absence of ubiquitylation-dependent degradation and deubiquitylation in E. coli significantly simplify the interpretation of the results. We harnessed the developed system to demonstrate that like NEDD4, UBE3B also undergoes self-ubiquitylation-dependent inactivation. We show that self-ubiquitylation of UBE3B on K665 induces oligomerization and inactivation in yeast and mammalian cells respectively. Finally, we showcase the advantages of split-CAT in the study of human diseases by demonstrating that mutations in UBE3B that cause Kaufman oculocerebrofacial syndrome exhibit clear E. coli growth phenotypes.

Using hippocampal-dependent eyeblink conditioning to predict individual differences in spatial reorientation strategies in 3- to 6-year-olds.

The hippocampus is a subcortical structure in the medial temporal lobe involved in cognitive functions such as spatial navigation and reorientation, episodic memory, and associative learning. While much is understood about the role of hippocampal function in learning and memory in adults, less is known about the relations between the hippocampus and the development of these cognitive skills in young children due to the limitations of using standard methods (e.g., MRI) to examine brain structure and function in developing populations. This study used hippocampal-dependent trace eyeblink conditioning (EBC) as a feasible approach to examine individual differences in hippocampal functioning as they relate to spatial reorientation and episodic memory performance in young children. Three- to six-year-old children (N = 50) completed tasks that measured EBC, spatial reorientation, and episodic memory, as well as non-hippocampal-dependent processing speed abilities. Results revealed that when age was held constant, individual differences in EBC performance were significantly related to individual differences in performance on the spatial reorientation test, but not on the episodic memory or processing speed tests. When the relations between hippocampal-dependent EBC and different reorientation strategies were explored, it was found that individual differences in hippocampal function predicted the use of geometric information for reorienting in space as opposed to a combined strategy that uses both geometric information and salient visual cues. The utilization of eyeblink conditioning to examine hippocampal function in young populations and its implications for understanding the dissociation between spatial reorientation and episodic memory development are discussed.

Language counts: Early language mediates the relationship between parent education and children's math ability.

Children's early math skills have been hailed as a powerful predictor of academic success. Disparities in socioeconomic context, however, also have dramatic consequences on children's learning. It is therefore critical to investigate both of these distinct contributors in order to better understand the early foundations of children's academic outcomes. This study tests an integrated model of children's developing math ability so as to (1) identify the specific skills and abilities most clearly linked to early math achievement and (2) measure the influence of children's socioeconomic context on each of these skills. We first evaluated the early vocabulary, number word knowledge (knower level), and Approximate Number System (ANS) acuity of a diverse group of preschoolers. Then, approximately 1 year later as they entered Kindergarten, we administered a test of early math achievement. We find that children's early language (general vocabulary and number word knowledge) fully mediates the relationship between parent education and math ability. Additionally, number word knowledge mediates the relationship between ANS acuity and early math. We argue that increased focus on number word knowledge, as well as general vocabulary, may help to minimize disparities in math ability as children enter kindergarten. We also highlight the role of parent education on children's learning and note that this may be an important locus for intervention.

E. coli-Based Selection and Expression Systems for Discovery, Characterization, and Purification of Ubiquitylated Proteins.

Ubiquitylation is an eukaryotic signal that regulates most cellular pathways. However, four major hurdles pose challenges to study ubiquitylation: (1) high redundancy between ubiquitin (Ub) cascades, (2) ubiquitylation is tightly regulated in the cell, (3) the transient nature of the Ub signal, and (4) difficulties to purify functional ubiquitylation apparatus for in vitro assay. Here, we present systems that express functional Ub cascades in E. coli, which lacks deubiquitylases, Ub-dependent degradations, and control mechanisms for ubiquitylation. Therefore, expression of an ubiquitylation cascade results in the accumulation of stable ubiquitylated protein that can be genetically selected or purified, thus circumventing the above challenges. Co-expression of split antibiotic resistance protein fragments tethered to Ub and ubiquitylation targets along with ubiquitylation enzymes (E1, E2, and E3) gives rise to bacterial growth on selective media. We show that ubiquitylation rate is highly correlated with growth efficiency. Hence, genetic libraries and simple manipulations in the selection system facilitate the identification and characterization of components and interfaces along Ub cascades. The bacterial expression system also facilitates the detection of ubiquitylated proteins. Furthermore, the expression system allows affinity chromatography-based purification of milligram quantities of ubiquitylated proteins for downstream biochemical, biophysical, and structural studies.

Do analog number representations underlie the meanings of young children's verbal numerals?

Children learn to count, and even learn the cardinal meanings of the first three or four verbal numerals ("one" through "three" or "four"), before they master the numerical significance of counting. If so, it follows that the cardinal meanings of those first few numerals cannot be derived, initially, from their place in the count list and the counting routine. What non-verbal representations, then, support the cardinal meanings of verbal numerals before children have mastered how counting does so? Four experiments addressed the commonly adopted assumption that in the earliest period of learning the meanings of number words, children map verbal numerals to regions of the analog number system (ANS), a system of representation with numerical content that is widely attested in animals and in human infants. Experiment 1 confirmed that children who know what "three" means, but who do not yet know what "four" means, and do not yet know how counting represents number, can be easily taught the meaning of "four," if they are trained to indicate sets of four when they are paired with a series of sets that contrast numerically with four. If children learn "four" by mapping the word to an ANS representation of sets of four, and if such ANS value-to-word mappings underlie the meanings of other known numerals early in development, then analogous teaching should enable young children to establish a ANS value-to-word mapping for between "ten" and sets of 10 as specified by the ANS. Furthermore, the ease of learning should be a function of the ratio of the number of individuals in the comparison set to 10. Three further experiments tested these hypotheses by attempting to teach young Cardinal Principle-knowers the meaning of the word "ten," under the same training conditions "three-"knowers are easily taught the meaning of "four". The children learned which picture in each training pair had "ten." However, test trials with novel animals and spatial configurations showed that they had failed to learn what set sizes should be labeled "ten", even when, after training, they were asked to indicate a set of 10 vs. a set of 20 or 30 (well within the ratio sensitivity of the ANS even early in infancy). Furthermore, there was no effect of ratio on success during test trials. These data provide new evidence that ANS value-to-word mappings do not underlie the meanings of number words early in development. We discuss what other non-verbal representations might do so, and discuss other ways the ANS may support learning how counting represents number.

A bacterial genetic selection system for ubiquitylation cascade discovery.

About one-third of the eukaryotic proteome undergoes ubiquitylation, but the enzymatic cascades leading to substrate modification are largely unknown. We present a genetic selection tool that utilizes Escherichia coli, which lack deubiquitylases, to identify interactions along ubiquitylation cascades. Coexpression of split antibiotic resistance protein tethered to ubiquitin and ubiquitylation target together with a functional ubiquitylation apparatus results in a covalent assembly of the resistance protein, giving rise to bacterial growth on selective media. We applied the selection system to uncover an E3 ligase from the pathogenic bacteria EHEC and to identify the epsin ENTH domain as an ultraweak ubiquitin-binding domain. The latter was complemented with a structure-function analysis of the ENTH-ubiquitin interface. We also constructed and screened a yeast fusion library, discovering Sem1 as a novel ubiquitylation substrate of Rsp5 E3 ligase. Collectively, our selection system provides a robust high-throughput approach for genetic studies of ubiquitylation cascades and for small-molecule modulator screening.

Frames of reference in spatial language acquisition.

Languages differ in how they encode spatial frames of reference. It is unknown how children acquire the particular frame-of-reference terms in their language (e.g., left/right, north/south). The present paper uses a word-learning paradigm to investigate 4-year-old English-speaking children's acquisition of such terms. In Part I, with five experiments, we contrasted children's acquisition of novel word pairs meaning left-right and north-south to examine their initial hypotheses and the relative ease of learning the meanings of these terms. Children interpreted ambiguous spatial terms as having environment-based meanings akin to north and south, and they readily learned and generalized north-south meanings. These studies provide the first direct evidence that children invoke geocentric representations in spatial language acquisition. However, the studies leave unanswered how children ultimately acquire "left" and "right." In Part II, with three more experiments, we investigated why children struggle to master body-based frame-of-reference words. Children successfully learned "left" and "right" when the novel words were systematically introduced on their own bodies and extended these words to novel (intrinsic and relative) uses; however, they had difficulty learning to talk about the left and right sides of a doll. This difficulty was paralleled in identifying the left and right sides of the doll in a non-linguistic memory task. In contrast, children had no difficulties learning to label the front and back sides of a doll. These studies begin to paint a detailed account of the acquisition of spatial terms in English, and provide insights into the origins of diverse spatial reference frames in the world's languages.

Acquisition of the Cardinal Principle Coincides with Improvement in Approximate Number System Acuity in Preschoolers.

Human mathematical abilities comprise both learned, symbolic representations of number and unlearned, non-symbolic evolutionarily primitive cognitive systems for representing quantities. However, the mechanisms by which our symbolic (verbal) number system becomes integrated with the non-symbolic (non-verbal) representations of approximate magnitude (supported by the Approximate Number System, or ANS) are not well understood. To explore this connection, forty-six children participated in a 6-month longitudinal study assessing verbal number knowledge and non-verbal numerical acuity. Cross-sectional analyses revealed a strong relationship between verbal number knowledge and ANS acuity. Longitudinal analyses suggested that increases in ANS acuity were most strongly related to the acquisition of the cardinal principle, but not to other milestones of verbal number acquisition. These findings suggest that experience with culture and language is intimately linked to changes in the properties of a core cognitive system.

Early group bias in the Faroe Islands: Cultural variation in children's group-based reasoning.

Recent developmental research demonstrates that group bias emerges early in childhood. However, little is known about the extent to which bias in minimal (i.e., arbitrarily assigned) groups varies with children's environment and experience, and whether such bias is universal across cultures. In this study, the development of group bias was investigated using a minimal groups paradigm with 46 four- to six-year-olds from the Faroe Islands. Children observed in-group and out-group members exhibiting varying degrees of prosocial behaviour (egalitarian or stingy sharing). Children did not prefer their in-group in the pretest, but a pro-in-group and anti-out-group sentiment emerged in both conditions in the posttest. Faroese children's response patterns differ from those of American children [Schug, M. G., Shusterman, A., Barth, H., & Patalano, A. L. (2013). Minimal-group membership influences children's responses to novel experience with group members. Developmental Science, 16(1), 47-55], suggesting that intergroup bias shows cultural variation even in a minimal groups context.

Into the pressure cooker: Student stress in college preparatory high schools.

PURPOSE: The goals of this study were to (1) measure psychological, physiological, and behavioral indicators of stress, (2) assess the relationship between stress and student attitudes, and (3) explore coping behaviors in response to stress, among a sample of students in two academically high-achieving environments. METHOD: Three hundred thirty-three students in grades 9 through 12 from two college-preparatory high schools completed a cross-sectional online survey that included the Students' Life Satisfaction Scale, School Attitude Assessment Questionnaire-Revised, and assessments for stress-related indicators, including eating, sleeping and exercise, and strategies they utilized for coping with stress. RESULTS: Students reported a high prevalence of physical and psychological correlates of stress, and related unhealthy behaviors such as widespread and chronic sleep deprivation and rushed meals. CONCLUSIONS: The results suggest areas to focus attention for identifying and addressing maladaptive responses to stress among high-achieving student populations.

The effect of friendly touch on delay-of-gratification in preschool children.

Physical touch has many documented benefits, but past research has paid little attention to the effects of touch on children's development. Here, we tested the effect of touch on children's compliance behaviour in a modified delay-of-gratification task. Forty children (M = 59 months) were randomly assigned to a touch or no touch group. Children in the intervention condition received a friendly touch on the back while being told that they should wait for permission to eat a candy. Results showed that children in the touch condition waited an average of two minutes longer to eat the candy than children in the no touch condition. This finding has implications for the potential of using touch to promote positive behaviours in children.

Minimal-group membership influences children's responses to novel experience with group members.

Children, like adults, tend to prefer ingroup over outgroup individuals, but how this group bias affects children's processing of information about social groups is not well understood. In this study, 5- and 6-year-old children were assigned to artificial groups. They observed instances of ingroup and outgroup members behaving in either a positive (egalitarian) or a negative (stingy) manner. Observations of positive ingroup and negative outgroup behaviors reliably reduced children's liking of novel outgroup members, while observations of negative ingroup and positive outgroup behaviors had little effect on liking ratings. In addition, children successfully identified the more generous group only when the ingroup was egalitarian and the outgroup stingy. These data provide compelling evidence that children treat knowledge of and experiences with ingroups and outgroups differently, and thereby differently interpret identical observations of ingroup versus outgroup members.

Cognitive effects of language on human navigation.

Language has been linked to spatial representation and behavior in humans, but the nature of this effect is debated. Here, we test whether simple verbal expressions improve 4-year-old children's performance in a disoriented search task in a small rectangular room with a single red landmark wall. Disoriented children's landmark-guided search for a hidden object was dramatically enhanced when the experimenter used certain verbal expressions to designate the landmark during the hiding event. Both a spatial expression ("I'm hiding the sticker at the red/white wall") and a non-spatial but task-relevant expression ("The red/white wall can help you get the sticker") enhanced children's search, relative to uncued controls. By contrast, a verbal expression that drew attention to the landmark in a task-irrelevant manner ("Look at this pretty red/white wall") produced no such enhancement. These findings provide further evidence that language changes spatial behavior in children and illuminate one mechanism through which language exerts its effect: by helping children understand the relevance of landmarks for encoding locations.

Evidence from an emerging sign language reveals that language supports spatial cognition.

Although spatial language and spatial cognition covary over development and across languages, determining the causal direction of this relationship presents a challenge. Here we show that mature human spatial cognition depends on the acquisition of specific aspects of spatial language. We tested two cohorts of deaf signers who acquired an emerging sign language in Nicaragua at the same age but during different time periods: the first cohort of signers acquired the language in its infancy, and 10 y later the second cohort of signers acquired the language in a more complex form. We found that the second-cohort signers, now in their 20s, used more consistent spatial language than the first-cohort signers, now in their 30s. Correspondingly, they outperformed the first cohort in spatially guided searches, both when they were disoriented and when an array was rotated. Consistent linguistic marking of left-right relations correlated with search performance under disorientation, whereas consistent marking of ground information correlated with search in rotated arrays. Human spatial cognition therefore is modulated by the acquisition of a rich language.