Rapid In Vitro Cytotoxicity Evaluation of Jurkat Expressing Chimeric Antigen Receptor using Fluorescent Imaging.

Chimeric antigen receptor (CAR) T cells are at the forefront of oncology. A CAR is constructed of a targeting domain (usually a single chain variable fragment, scFv), with an accompanying intra-chain linker, followed by a hinge, transmembrane, and costimulatory domain. Modification of the intra-chain linker and hinge domain can have a significant effect on CAR-mediated killing. Considering the many different options for each part of a CAR construct, there are large numbers of permutations. Making CAR-T cells is a time-consuming and expensive process, and making and testing many constructs is a heavy time and material investment. This protocol describes a platform to rapidly evaluate hinge-optimized CAR constructs in Jurkat cells (CAR-J). Jurkat cells are an immortalized T cell line with high lentivirus uptake, allowing for efficient CAR transduction. Here, we present a platform to rapidly evaluate CAR-J using a fluorescent imager, followed by confirmation of cytolysis in PBMC-derived T cells.

Inducing Mitotic Catastrophe as a Therapeutic Approach to Improve Outcomes in Ewing Sarcoma.

Ewing sarcoma (EWS) is an aggressive pediatric malignancy of the bone and soft tissues in need of novel therapeutic options. To identify potential therapeutic targets, we focused on essential biological pathways that are upregulated by EWS-FLI1, the primary oncogenic driver of EWS, including mitotic proteins such as Aurora kinase A (AURKA) and kinesin family member 15 (KIF15) and its binding partner, targeting protein for Xklp2 (TPX2). KIF15/TPX2 cooperates with KIF11, a key mitotic kinesin essential for mitotic spindle orientation. Given the lack of clinical-grade KIF15/TPX2 inhibitors, we chose to target KIF11 (using SB-743921) in combination with AURKA (using VIC-1911) given that phosphorylation of KIF15S1169 by Aurora A is required for its targeting to the spindle. In vitro, the drug combination demonstrated strong synergy (Bliss score ≥ 10) at nanomolar doses. Colony formation assay revealed significant reduction in plating efficiency (1-3%) and increased percentage accumulation of cells in the G2/M phase with the combination treatment (45-52%) upon cell cycle analysis, indicating mitotic arrest. In vivo studies in EWS xenograft mouse models showed significant tumor reduction and overall effectiveness: drug combination vs. vehicle control (p ≤ 0.01), SB-743921 (p ≤ 0.01) and VIC-1911 (p ≤ 0.05). Kaplan-Meier curves demonstrated superior overall survival with the combination compared to vehicle or monotherapy arms (p ≤ 0.0001).

High-throughput and targeted drug screens identify pharmacological candidates against MiT-translocation renal cell carcinoma.

BACKGROUND: MiT-Renal Cell Carcinoma (RCC) is characterized by genomic translocations involving microphthalmia-associated transcription factor (MiT) family members TFE3, TFEB, or MITF. MiT-RCC represents a specific subtype of sporadic RCC that is predominantly seen in young patients and can present with heterogeneous histological features making diagnosis challenging. Moreover, the disease biology of this aggressive cancer is poorly understood and there is no accepted standard of care therapy for patients with advanced disease. Tumor-derived cell lines have been established from human TFE3-RCC providing useful models for preclinical studies. METHODS: TFE3-RCC tumor derived cell lines and their tissues of origin were characterized by IHC and gene expression analyses. An unbiased high-throughput drug screen was performed to identify novel therapeutic agents for treatment of MiT-RCC. Potential therapeutic candidates were validated in in vitro and in vivo preclinical studies. Mechanistic assays were conducted to confirm the on-target effects of drugs. RESULTS: The results of a high-throughput small molecule drug screen utilizing three TFE3-RCC tumor-derived cell lines identified five classes of agents with potential pharmacological efficacy, including inhibitors of phosphoinositide-3-kinase (PI3K) and mechanistic target of rapamycin (mTOR), and several additional agents, including the transcription inhibitor Mithramycin A. Upregulation of the cell surface marker GPNMB, a specific MiT transcriptional target, was confirmed in TFE3-RCC and evaluated as a therapeutic target using the GPNMB-targeted antibody-drug conjugate CDX-011. In vitro and in vivo preclinical studies demonstrated efficacy of the PI3K/mTOR inhibitor NVP-BGT226, Mithramycin A, and CDX-011 as potential therapeutic options for treating advanced MiT-RCC as single agents or in combination. CONCLUSIONS: The results of the high-throughput drug screen and validation studies in TFE3-RCC tumor-derived cell lines have provided in vitro and in vivo preclinical data supporting the efficacy of the PI3K/mTOR inhibitor NVP-BGT226, the transcription inhibitor Mithramycin A, and GPNMB-targeted antibody-drug conjugate CDX-011 as potential therapeutic options for treating advanced MiT-RCC. The findings presented here should provide the basis for designing future clinical trials for patients with MiT-driven RCC.

"It Happened When I Was Connecting to the Community ": Multiple Pathways to Migrant (Non)Belonging in a New Destination Setting.

Migrants' sense of belonging in their country and community of residence has direct effects on their health and wellbeing. A diverse set of case studies suggest that legal immigration status plays a primary role in shaping migrants' opportunities for and experiences of belonging. Few of these studies, though, have examined belonging for migrants with varied legal immigration statuses living in the same receiving context, limiting our understanding of if and how migrant status interacts with other factors to shape access to belonging for migrants settling in the same host community. To address this gap, we analyze 73 semi-structured interviews with migrants in Utah, USA, to investigate the process and experience of belonging for migrants across permanent, temporary, undocumented, and refugee statuses. While legal immigration status is an important factor shaping (non)belonging, it does not appear to function as a master status for migrant belonging. Rather, we find that legal immigration status works alongside a number of community-level factors-including cultural, social, linguistic, and racial/ethnic factors-to shape belonging for migrants of all immigration statuses. These non-legal, community-level factors emerged as critical features of (non)belonging for many migrants living in Utah. Our findings suggest that, although they cannot change federal immigration policies, local- and state-level governments and organizations can enhance migrants' access to belonging and wellbeing across many other dimensions.

The Academic Cost of Worry Among Socioeconomically Disadvantaged Children.

OBJECTIVES: Worry and loneliness looms large in American schools, especially in the social years of early adolescence where friendships are in flux and children strive to fit in and do well academically. We examine a nationally-representative sample of American 5th graders to document the extent of academic worry and loneliness, its costs for academic performance, and how social class can disrupt or exacerbate its associations. METHODS: Based on a nationally representative longitudinal survey (ECLS-K 2010-2011) of childhood (N = 5750), we examine if a child's self-reported worry and loneliness are associated with standardized math and reading scores using OLS regression. We explore whether these associations vary by socioeconomic status. RESULTS: We find that academic worry is a strong predictor of math and reading skill. The association is amplified for disadvantaged students. Patterns hold when accounting for a host of other factors and are replicated in the ECLS-K 1998-1999. Loneliness and its association with math and reading performance was not statistically significant. CONCLUSIONS FOR PRACTICE: As academic worry is negatively associated with standardized math and reading skills, practitioners can be especially attuned to how these patterns are amplified for children in low socioeconomic households. Utilizing a nationally representative survey of early adolescence, we show that worry (and less so loneliness) is associated with math and reading skills and that these associations are moderated by socioeconomic status-disadvantaged students have a higher negative association with math and reading performance when they worry about their academic performance compared to advantaged students.

Early adverse childhood experiences and exclusionary discipline in high school.

The use of school suspension and expulsion is a widespread phenomenon in American schools (Wallace et al., 2009; Owens and McLanahan, 2020). Yet, much of what we know about these exclusionary practices provide little insight into the personal biographies of the students themselves-specifically their histories of childhood trauma. Using measures of adverse childhood experiences (ACEs), we examine the link between early ACEs (up to age 5) and school suspension/expulsion using the Fragile Families and Child Wellbeing Study (1998-2010) (FFCWS). We find that a child with a cumulative ACE score are almost four times more likely to have been suspended or expelled. Importantly, this negative link persists even when accounting for factors known to be associated with ACEs and school discipline. This work offers new theoretical insight into how we understand discipline in school contexts and suggests the importance of trauma-informed interventions in the American education system.

Hypoxic preconditioning protects against ischemic kidney injury through the IDO1/kynurenine pathway.

Prolonged cellular hypoxia leads to energetic failure and death. However, sublethal hypoxia can trigger an adaptive response called hypoxic preconditioning. While prolyl-hydroxylase (PHD) enzymes and hypoxia-inducible factors (HIFs) have been identified as key elements of oxygen-sensing machinery, the mechanisms by which hypoxic preconditioning protects against insults remain unclear. Here, we perform serum metabolomic profiling to assess alterations induced by two potent cytoprotective approaches, hypoxic preconditioning and pharmacologic PHD inhibition. We discover that both approaches increase serum kynurenine levels and enhance kynurenine biotransformation, leading to preservation of NAD+ in the post-ischemic kidney. Furthermore, we show that indoleamine 2,3-dioxygenase 1 (Ido1) deficiency abolishes the systemic increase of kynurenine and the subsequent renoprotection generated by hypoxic preconditioning and PHD inhibition. Importantly, exogenous administration of kynurenine restores the hypoxic preconditioning in the context of Ido1 deficiency. Collectively, our findings demonstrate a critical role of the IDO1-kynurenine axis in mediating hypoxic preconditioning.

Development and initial clinical testing of a multiplexed circulating tumor cell assay in patients with clear cell renal cell carcinoma.

Although therapeutic options for patients with advanced renal cell carcinoma (RCC) have increased in the past decade, no biomarkers are yet available for patient stratification or evaluation of therapy resistance. Given the dynamic and heterogeneous nature of clear cell RCC (ccRCC), tumor biopsies provide limited clinical utility, but liquid biopsies could overcome these limitations. Prior liquid biopsy approaches have lacked clinically relevant detection rates for patients with ccRCC. This study employed ccRCC-specific markers, CAIX and CAXII, to identify circulating tumor cells (CTC) from patients with metastatic ccRCC. Distinct subtypes of ccRCC CTCs were evaluated for PD-L1 and HLA-I expression and correlated with patient response to therapy. CTC enumeration and expression of PD-L1 and HLA-I correlated with disease progression and treatment response, respectively. Longitudinal evaluation of a subset of patients demonstrated potential for CTC enumeration to serve as a pharmacodynamic biomarker. Further evaluation of phenotypic heterogeneity among CTCs is needed to better understand the clinical utility of this new biomarker.

Female Sex Offenders: Is There a Difference Between Solo and Co-Offenders?

Studies on female sex offending have been limited for a number of reasons, such as societal perceptions that females are incapable of engaging in such behaviors because of their role as caretakers and nurturers in society. However, over the past few decades, studies examining female sex offenders have increased, revealing that females do commit sexual offenses and differ from their male counterparts. We examined offender, victim, and offense characteristics of female sex offenders who were convicted from 1995 to 2013 (N = 223) in Arkansas and were sentenced to serve time in prison or placed on probation. We focused on the similarities and differences of solo and co-female sex offenders because we know from previous studies that the pathway of offending can differ between solo and co-female offenders, yet few studies have exclusively compared the similarities and differences among female sex offenders. Our data were collected from offender files that included basic personal offender information, offender survey and social history, criminal history, incident reports while incarcerated, court records, police investigation reports, initial offender and victim statements (prior to offender incarceration), and probation/parole reports. We believe the results of this study will provide further insight into the types of female sex offenders as well as the possible differences between co- and solo-offenders in relation to their victim preferences, risk levels, rehabilitation amenability, and recidivism propensities.

Targeting loss of the Hippo signaling pathway in NF2-deficient papillary kidney cancers.

Papillary renal cell carcinomas (PRCC) are a histologically and genetically heterogeneous group of tumors that represent 15-20% of all kidney neoplasms and may require diverse therapeutic approaches. Alteration of the NF2 tumor suppressor gene, encoding a key regulator of the Hippo signaling pathway, is observed in 22.5% of PRCC. The Hippo signaling pathway controls cell proliferation by regulating the transcriptional activity of Yes-Associated Protein, YAP1. Loss of NF2 results in aberrant YAP1 activation. The Src family kinase member Yes also regulates YAP1 transcriptional activity. This study investigated the importance of YAP and Yes activity in three NF2-deficient PRCC cell lines. NF2-deficency correlated with increased expression of YAP1 transcriptional targets and siRNA-based knockdown of YAP1 and Yes1 downregulated this pathway and dramatically reduced cell viability. Dasatinib and saracatinib have potent inhibitory effects on Yes and treatment with either resulted in downregulation of YAP1 transcription targets, reduced cell viability, and G0-G1 cell cycle arrest. Xenograft models for NF2-deficient PRCC also demonstrated reduced tumor growth in response to dasatinib. Thus, inhibiting Yes and the subsequent transcriptional activity of YAP1 had a substantial anti-tumor cell effect both in vitro and in vivo and may provide a viable therapeutic approach for patients with NF2-deficient PRCC.

Breastfeeding, Parenting, and Infant Attachment Behaviors.

Objectives Infants and toddlers need secure attachments in order to develop the social competence required to successfully navigate later peer and adult relationships. Breastfeeding is a parenting factor that has been associated with child emotional development-specifically the attachment between children and their mothers. Yet, this link may simply be the result of other parenting behaviors that are associated with breastfeeding. Thus, our objective is to examine whether the link between infant attachment behaviors and breastfeeding endures when accounting for a broad array of in-depth measures of parenting. Methods We use the Early Childhood Longitudinal Study of children from 9 months to 2 years of age collected by the National Center for Education Statistics. Using Ordinary Least Squares regression, data analyses examine the association between the Toddler Attachment Sort-45 (TAS-45) measures of toddler-parent attachment (infant attachment security and temperamental dependency) and breastfeeding practices. We also examine individual items of the TAS-45 to isolate specific attachment behaviors that have the strongest associations with breastfeeding. Results We find an enduring link between children who are predominantly breastfed for six or more months and infant attachment security. However, we find no evidence that breastfeeding is linked to a child's temperamental dependency. Of the nine items used to examine infant attachment behaviors, we find that breastfed children are rated as having slightly higher scores on two measures ("warm and cuddly," "cooperative") and lower scores on one measure ("demanding/angry"). Conclusions for Practice Breastfeeding has an important link to the child's use of their caregiver as a secure base for exploration and a place of comfort when distressed (infant attachment security). Yet, breastfeeding does not appear to reduce a child's temperamental dependency or level of clinginess as measured by how demanding, fussy or distressed the child becomes when separated.

Detecting the Potential Pharmacological Synergy of Drug Combination by Viability Assays In Vitro.

Heat shock protein 90 (HSP90) is a molecular chaperone necessary for the folding and proper function of multiple "client" proteins. HSP90 is involved in numerous biological processes and is critical to maintain proteostasis and to protect the cells from potentially harmful environmental stresses such as heat. However, in cancer, the role of HSP90, and other molecular chaperones, is corrupted as many of HSP90 clients are kinases and transcription factors whose aberrant activation or mutation drives tumor growth. Thus, developing a polytherapy, or combination therapy, that includes an HSP90 inhibitor in addition to targeting an oncogene or oncogenic pathway is an appealing therapeutic approach. This protocol will provide detailed methods on how to assess the potential synergy of polytherapy by viability assays in vitro.

The relative age effect reversal among the National Hockey League elite.

Like many sports in adolescence, junior hockey is organized by age groups. Typically, players born after December 31st are placed in the subsequent age cohort and as a result, will have an age advantage over those players born closer to the end of the year. While this relative age effect (RAE) has been well-established in junior hockey and other professional sports, the long-term impact of this phenomenon is not well understood. Using roster data on North American National Hockey League (NHL) players from the 2008-2009 season to the 2015-2016 season, we document a RAE reversal-players born in the last quarter of the year (October-December) score more and command higher salaries than those born in the first quarter of the year. This reversal is even more pronounced among the NHL "elite." We find that among players in the 90th percentile of scoring, those born in the last quarter of the year score about 9 more points per season than those born in the first quarter. Likewise, elite players in the 90th percentile of salary who are born in the last quarter of the year earn 51% more pay than players born at the start of the year. Surprisingly, compared to players at the lower end of the performance distribution, the RAE reversal is about three to four times greater among elite players.

Discovering Targets of Non-enzymatic Acylation by Thioester Reactivity Profiling.

Non-enzymatic protein modification driven by thioester reactivity is thought to play a major role in the establishment of cellular lysine acylation. However, the specific protein targets of this process are largely unknown. Here we report an experimental strategy to investigate non-enzymatic acylation in cells. Specifically, we develop a chemoproteomic method that separates thioester reactivity from enzymatic utilization, allowing selective enrichment of non-enzymatic acylation targets. Applying this method to cancer cell lines identifies numerous candidate targets of non-enzymatic acylation, including several enzymes in lower glycolysis. Functional studies highlight malonyl-CoA as a reactive thioester metabolite that can modify and inhibit glycolytic enzyme activity. Finally, we show that synthetic thioesters can be used as novel reagents to probe non-enzymatic acylation in living cells. Our studies provide new insights into the targets and drivers of non-enzymatic acylation, and demonstrate the utility of reactivity-based methods to experimentally investigate this phenomenon in biology and disease.

Integrated Analysis of Multiple Biomarkers from Circulating Tumor Cells Enabled by Exclusion-Based Analyte Isolation.

PURPOSE: There is a critical clinical need for new predictive and pharmacodynamic biomarkers that evaluate pathway activity in patients treated with targeted therapies. A microscale platform known as VERSA (versatile exclusion-based rare sample analysis) was developed to integrate readouts across protein, mRNA, and DNA in circulating tumor cells (CTC) for a comprehensive analysis of the androgen receptor (AR) signaling pathway. EXPERIMENTAL DESIGN: Utilizing exclusion-based sample preparation principles, a handheld chip was developed to perform CTC capture, enumeration, quantification, and subcellular localization of proteins and extraction of mRNA and DNA. This technology was validated across integrated endpoints in cell lines and a cohort of patients with castrate-resistant prostate cancer (CRPC) treated with AR-targeted therapies and chemotherapies. RESULTS: The VERSA was validated in cell lines to analyze AR protein expression, nuclear localization, and gene expression targets. When applied to a cohort of patients, radiographic progression was predicted by the presence of multiple AR splice variants and activity in the canonical AR signaling pathway. AR protein expression and nuclear localization identified phenotypic heterogeneity. Next-generation sequencing with the FoundationOne panel detected copy number changes and point mutations. Longitudinal analysis of CTCs identified acquisition of multiple AR variants during targeted treatments and chemotherapy. CONCLUSIONS: Complex mechanisms of resistance to AR-targeted therapies, across RNA, DNA, and protein endpoints, exist in patients with CRPC and can be quantified in CTCs. Interrogation of the AR signaling pathway revealed distinct patterns relevant to tumor progression and can serve as pharmacodynamic biomarkers for targeted therapies. Clin Cancer Res; 1-11. ©2016 AACR.

Birth Weight and Early Cognitive Skills: Can Parenting Offset the Link?

Objectives There is an enduring negative association between low birth weight (<2500 g) and early childhood cognitive skills. This study examines if parenting practices meaningfully contribute to or offset birth weight disparities in cognitive development prior to formal schooling. Methods This study uses the ECLS-B, a nationally representative sample of live births in the United States in 2001. Unlike studies focused on one or two measures of parenting and investment, this study considers a wide array parenting measures collected at multiple time points, tracked from before birth across 5 years of development. Results Regression results show that nearly 50 % of the low-birth-weight gap in early math and reading ability is associated with family socioeconomic status. Between-family OLS regressions show that parenting practices, including "parental interaction," "cognitive stimulation," and "parent quality", are negatively associated with low birth weight and positively associated with improved cognitive skill among all children. After adjustment for family socioeconomic status, parenting practices did little to offset (by mediation or moderation) remaining birth weight disparities in early cognitive development. Conclusions Effective parenting is positively associated with cognitive development, but parenting is not a panacea-the developmental disadvantages associated with poor child health are not linked to parenting practices. We argue that birth weight disparities are rooted in biology and cannot easily be offset by parenting practices.

The (Conditional) Resource Dilution Model: State- and Community-Level Modifications.

One of the most consistent patterns in the social sciences is the relationship between sibship size and educational outcomes: those with fewer siblings outperform those with many. The resource dilution (RD) model emphasizes the increasing division of parental resources within the nuclear family as the number of children grows, yet it fails to account for instances when the relationship between sibship size and education is often weak or even positive. To reconcile, we introduce a conditional resource dilution (CRD) model to acknowledge that nonparental investments might aid in children's development and condition the effect of siblings. We revisit the General Social Surveys (1972-2010) and find support for a CRD approach: the relationship between sibship size and educational attainment has declined during the first half of the twentieth century, and this relationship varies across religious groups. Findings suggest that state and community resources can offset the impact of resource dilution-a more sociological interpretation of sibship size patterns than that of the traditional RD model.

Extracurricular associations and college enrollment.

There is consistent evidence that student involvement in extracurricular activities (EAs) is associated with numerous academic benefits, yet understanding how peer associations within EAs might influence this link is not well understood. Using Add Health's comprehensive data on EA participation across 80 schools in the United States, we develop a novel measure of peer associations within EA activities. We find that EA participation with high achieving peers has a nontrivial link to college enrollment, even after considering individual, peer, and school-level factors. This suggests that school policies aimed at encouraging student exposure to high achieving peers in EAs could have an important impact on a student's later educational outcomes.

Breastfeeding, parenting, and early cognitive development.

OBJECTIVE: To explain why breastfeeding is associated with children's cognitive development. STUDY DESIGN: By using a nationally representative longitudinal survey of early childhood (N = 7500), we examined how breastfeeding practices, the early introduction of solid foods, and putting an infant to bed with a bottle were associated with cognitive development across early childhood. We also explored whether this link can be explained by parenting behaviors and maternal education. RESULTS: There is a positive relationship between predominant breastfeeding for 3 months or more and child reading skills, but this link is the result of cognitively supportive parenting behaviors and greater levels of education among women who predominantly breastfed. We found little-to-no relationship between infant feeding practices and the cognitive development of children with less-educated mothers. Instead, reading to a child every day and being sensitive to a child's development were significant predictors of math and reading readiness outcomes. CONCLUSIONS: Although breastfeeding has important benefits in other settings, the encouragement of breastfeeding to promote school readiness does not appear to be a key intervention point. Promoting parenting behaviors that improve child cognitive development may be a more effective and direct strategy for practitioners to adopt, especially for disadvantaged children.