A Non-Nuclear NF-κB Modulates Alcohol Sensitivity But Not Immunity.

NF-κB proteins are well known as transcription factors important in immune system activation. In this highly conserved role, they contribute to changes in behavior in response to infection and in response to a variety of other insults and experiences. In some mammalian neurons, NF-κBs can be found at the synapse and translocate to the nucleus to alter gene expression when activated by synaptic activity. Here, we demonstrate that, in Drosophila melanogaster, NF-κB action is important both inside and outside the nucleus and that the Dif gene has segregated nuclear and non-nuclear NF-κB action into different protein isoforms. The DifA isoform is a canonical nuclear-acting NF-κB protein that enters the nucleus and is important for combating infection. The DifB variant, but not the DifA variant, is found in the central nervous system (mushroom bodies and antennal lobes). DifB does not enter the nucleus and co-localizes with a synaptic protein. In males and females, a DifB mutant alters alcohol behavioral sensitivity without an obvious effect on combating infection, whereas a DifA mutant does not affect alcohol sensitivity but compromises the immune response. These data are evidence that the non-nuclear DifB variant contributes to alcohol behavioral sensitivity by a nongenomic mechanism that diverges from the NF-κB transcriptional effects used in the peripheral immune system. Enrichment of DifB in brain regions rich in synapses and biochemical enrichment of DifB in the synaptoneurosome fraction indicates that the protein may act locally at the synapse.SIGNIFICANCE STATEMENT NF-κBs are transcription factors used by innate immune signaling pathways to protect against infection. Alcohol abuse also activates these pathways, which contributes to the addictive process and the health consequences associated with alcohol abuse. In the mammalian nervous system, NF-κBs localize to synapses, but it is axiomatic that they effect change by acting in the nucleus. However, for the Drosophila Dif gene, immune and neural function segregate into different protein isoforms. Whereas the nuclear isoform (DifA) activates immune genes in response to infection, the CNS isoform acts nongenomically to modulate alcohol sensitivity. Immunohistochemical and biochemical assays localize DifB to synapse-rich regions. Direct synaptic action would provide a novel and rapid way for NF-κB signaling to modulate behavior.

Translatability of In Vitro Stress for Predicting Deamidation and Oxidation Biotransformation on Biotherapeutics.

Biotransformation leading to single residue modifications (e.g., deamidation, oxidation) can contribute to decreased efficacy/potency, poor pharmacokinetics, and/or toxicity/immunogenicity for protein therapeutics. Identifying and characterizing such liabilities in vivo are emerging needs for biologics drug discovery. In vitro stress assays involving PBS for deamidation or AAPH for oxidation are commonly used for predicting liabilities in manufacturing and storage and are sometimes considered a predictive tool for in vivo liabilities. However, reports discussing their in vivo translatability are limited. Herein, we introduce a mass spectrometry workflow that characterizes in vivo oxidation and deamidation in pharmacokinetically relevant compartments for diverse protein therapeutic modalities. The workflow has low bias of <10% in quantitating degradation in the relevant pharmacokinetic concentration range for monkey and rabbit serum/plasma (1-100 µg/mL) and allows for high sequence coverage (∼85%) for discovery/monitoring of amino acid modifications. For oxidation and deamidation, the assay was precise, with percent coefficient of variation of <8% at 1-100 µg/mL and ≤6% method-induced artifacts. A high degree of in vitro and in vivo correlation was observed for deamidation on the six diverse protein therapeutics (seven liability sites) tested. In vivo translatability for oxidation liabilities were not observed for the 11 molecules tested using in vitro AAPH stress. One of the molecules dosed in eyes resulted in a false positive and a false negative prediction for in vivo oxidation following AAPH stress. Finally, peroxide stress was also tested but resulted in limited success (1 out of 4 molecules) in predicting oxidation liabilities.

Structure of Deep Eutectic Solvents (DESs): What We Know, What We Want to Know, and Why We Need to Know It.

Deep eutectic solvents (DESs) are a tunable class of solvents with many advantageous properties including good thermal stability, facile synthesis, low vapor pressure, and low-to-negligible toxicity. DESs are composed of hydrogen bond donors and acceptors that, when combined, significantly decrease the freezing point of the resulting solvent. DESs have distinct interfacial and bulk structural heterogeneity compared to traditional solvents, in part due to various intramolecular and intermolecular interactions. Many of the physiochemical properties observed for DESs are influenced by structure. However, our understanding of the interfacial and bulk structure of DESs is incomplete. To fully exploit these solvents in a range of applications including catalysis, separations, and electrochemistry, a better understanding of DES structure must be obtained. In this Perspective, we provide an overview of the current knowledge of the interfacial and bulk structure of DESs and suggest future research directions to improve our understanding of this important information.

Skin barrier lipid enzyme activity in Netherton patients is associated with protease activity and ceramide abnormalities.

Individuals with Netherton syndrome (NTS) have increased serine protease activity, which strongly impacts the barrier function of the skin epidermis and leads to skin inflammation. Here, we investigated how serine protease activity in NTS correlates with changes in the stratum corneum (SC) ceramides, which are crucial components of the skin barrier. We examined two key enzymes involved in epidermal ceramide biosynthesis, ß-glucocerebrosidase (GBA) and acid-sphingomyelinase (ASM). We compared in situ expression levels and activities of GBA and ASM between NTS patients and controls and correlated the expression and activities with i) SC ceramide profiles, ii) in situ serine protease activity, and iii) clinical presentation of patients. Using activity-based probe labeling, we visualized and localized active epidermal GBA, and a newly developed in situ zymography method enabled us to visualize and localize active ASM. Reduction in active GBA in NTS patients coincided with increased ASM activity, particularly in areas with increased serine protease activity. NTS patients with scaly erythroderma exhibited more pronounced anomalies in GBA and ASM activities than patients with ichthyosis linearis circumflexa. They also displayed a stronger increase in SC ceramides processed via ASM. We conclude that changes in the localization of active GBA and ASM correlate with i) altered SC ceramide composition in NTS patients, ii) local serine protease activity, and iii) the clinical manifestation of NTS.

Cryptococcosis in pregnancy and the postpartum period: Case series and systematic review with recommendations for management.

Cryptococcal meningitis causes 15% of AIDS-related deaths. Optimal management and clinical outcomes of pregnant women with cryptococcosis are limited to case reports, as pregnant women are often excluded from research. Amongst pregnant women with asymptomatic cryptococcosis, no treatment guidelines exist. We prospectively identified HIV-infected women who were pregnant or recently pregnant with cryptococcosis, screened during a series of meningitis research studies in Uganda from 2012 to 2018. Among 571 women screened for cryptococcosis, 13 were pregnant, one was breastfeeding, three were within 14 days postpartum, and two had recently miscarried. Of these 19 women (3.3%), 12 had cryptococcal meningitis, six had cryptococcal antigenemia, and one had a history of cryptococcal meningitis and was receiving secondary prophylaxis. All women with meningitis received amphotericin B deoxycholate (0.7-1.0 mg/kg). Five were exposed to 200-800 mg fluconazole during pregnancy. Of these five, three delivered healthy babies with no gross physical abnormalities at birth, one succumbed to meningitis, and one outcome was unknown. Maternal meningitis survival rate at hospital discharge was 75% (9/12), and neonatal/fetal survival rate was 44% (4/9) for those mothers who survived. Miscarriages and stillbirths were common (n = 4). Of six women with cryptococcal antigenemia, two received fluconazole, one received weekly amphotericin B, and three had unknown treatment courses. All women with antigenemia survived, and none developed clinical meningitis. We report good maternal outcomes but poor fetal outcomes for cryptococcal meningitis using amphotericin B, without fluconazole in the first trimester, and weekly amphotericin B in place of fluconazole for cryptococcal antigenemia.

A Difference-Education Intervention Equips First-Generation College Students to Thrive in the Face of Stressful College Situations.

A growing social psychological literature reveals that brief interventions can benefit disadvantaged students. We tested a key component of the theoretical assumption that interventions exert long-term effects because they initiate recursive processes. Focusing on how interventions alter students' responses to specific situations over time, we conducted a follow-up lab study with students who had participated in a difference-education intervention 2 years earlier. In the intervention, students learned how their social-class backgrounds mattered in college. The follow-up study assessed participants' behavioral and hormonal responses to stressful college situations. We found that difference-education participants discussed their backgrounds in a speech more frequently than control participants did, an indication that they retained the understanding of how their backgrounds mattered. Moreover, among first-generation students (i.e., students whose parents did not have 4-year degrees), those in the difference-education condition showed greater physiological thriving (i.e., anabolic-balance reactivity) than those in the control condition, which suggests that they experienced their working-class backgrounds as a strength.

Social class culture cycles: how three gateway contexts shape selves and fuel inequality.

America's unprecedented levels of inequality have far-reaching negative consequences for society as a whole. Although differential access to resources contributes to inequality, the current review illuminates how ongoing participation in different social class contexts also gives rise to culture-specific selves and patterns of thinking, feeling, and acting. We integrate a growing body of interdisciplinary research to reveal how social class culture cycles operate over the course of the lifespan and through critical gateway contexts, including homes, schools, and workplaces. We first document how each of these contexts socializes social class cultural differences. Then, we demonstrate how these gateway institutions, which could provide access to upward social mobility, are structured according to middle-class ways of being a self and thus can fuel and perpetuate inequality. We conclude with a discussion of intervention opportunities that can reduce inequality by taking into account the contextual responsiveness of the self.

Closing the social-class achievement gap: a difference-education intervention improves first-generation students' academic performance and all students' college transition.

College students who do not have parents with 4-year degrees (first-generation students) earn lower grades and encounter more obstacles to success than do students who have at least one parent with a 4-year degree (continuing-generation students). In the study reported here, we tested a novel intervention designed to reduce this social-class achievement gap with a randomized controlled trial (N = 168). Using senior college students' real-life stories, we conducted a difference-education intervention with incoming students about how their diverse backgrounds can shape what they experience in college. Compared with a standard intervention that provided similar stories of college adjustment without highlighting students' different backgrounds, the difference-education intervention eliminated the social-class achievement gap by increasing first-generation students' tendency to seek out college resources (e.g., meeting with professors) and, in turn, improving their end-of-year grade point averages. The difference-education intervention also improved the college transition for all students on numerous psychosocial outcomes (e.g., mental health and engagement).

Interdependent behavior only benefits employees from working-class backgrounds when it is both enacted and valued.

Social class disparities are pervasive in American society. In higher education, one critical driver of these disparities is the cultural mismatch between the interdependent norms of people from working-class backgrounds and the independent norms that pervade higher education. However, after graduating from college and entering white-collar workplaces, people from working-class backgrounds have frequent opportunities to collaborate in teams-that is, to enact interdependent behavior. Do these opportunities reduce cultural mismatch for people from working-class backgrounds? Across two survey studies and two experiments with college-educated U.S. employees (total N = 2,566), we find that they do not. We theorize and document that this is because there is often a decoupling between enacting interdependent behavior and whether such behavior is valued as part of being a "good" employee. We find that employees from working-class backgrounds only experience a cultural match and its benefits (e.g., sense of fit, high retention intentions) when interdependent behaviors are both enacted and valued. In contrast, when interdependent behaviors are enacted but not valued, employees from working-class backgrounds experience a cultural mismatch. Furthermore, we find that this pattern is unique to employees from working-class backgrounds: Employees from middle-class backgrounds report similar fit and retention regardless of whether there is a coupling of enacted and valued interdependent behavior. Taken together, our results suggest that it is critical to examine multiple elements of culture simultaneously (e.g., both enacted and valued behavior) to fully understand and predict the consequences of cultural (mis)match. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

The benefits of difference-education interventions in lower-resourced institutions.

Difference-education is an intervention that addresses psychological barriers that can undermine the academic performance of first-generation college students (i.e., those who have parents without 4-year degrees). Difference-education interventions improve first-generation students' performance by empowering them to navigate higher education environments more effectively. They also improve students' comfort with social group difference. However, these benefits have only been documented in higher-resourced institutions. The present research asks two questions about whether these benefits also extend to lower-resourced institutions-that is, schools with fewer resources to invest in students than the universities where prior difference-education interventions were delivered. First, is difference-education effective in improving first-generation students' academic performance in lower-resourced institutions, and does it do so by increasing empowerment? Second, does difference-education improve comfort with social group difference in lower-resourced institutions, and is it unique in its ability to do so? With students from four lower-resourced institutions, we examined these questions by comparing the results of a difference-education intervention to a control condition and social-belonging intervention. We found that while some benefits of difference-education interventions extend to lower-resourced institutions, others do not. First, like prior interventions, difference-education improves first-generation students' academic performance and comfort with social group difference. Unlike prior interventions, these effects did not persist beyond the first term and students' academic performance benefits were not explained by empowerment. We also found partial evidence that the benefits for comfort with social group difference were unique compared to a social-belonging intervention. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Structure- and machine learning-guided engineering demonstrate that a non-canonical disulfide in an anti-PD-1 rabbit antibody does not impede antibody developability.

Rabbits produce robust antibody responses and have unique features in their antibody repertoire that make them an attractive alternative to rodents for in vivo discovery. However, the frequent occurrence of a non-canonical disulfide bond between complementarity-determining region (CDR) H1 (C35a) and CDRH2 (C50) is often seen as a liability for therapeutic antibody development, despite limited reports of its effect on antibody binding, function, and stability. Here, we describe the discovery and humanization of a human-mouse cross-reactive anti-programmed cell death 1 (PD-1) monoclonal rabbit antibody, termed h1340.CC, which possesses this non-canonical disulfide bond. Initial removal of the non-canonical disulfide resulted in a loss of PD-1 affinity and cross-reactivity, which led us to explore protein engineering approaches to recover these. First, guided by the sequence of a related clone and the crystal structure of h1340.CC in complex with PD-1, we generated variant h1340.SA.LV with a potency and cross-reactivity similar to h1340.CC, but only partially recovered affinity. Side-by-side developability assessment of both h1340.CC and h1340.SA.LV indicate that they possess similar, favorable properties. Next, and prompted by recent developments in machine learning (ML)-guided protein engineering, we used an unbiased ML- and structure-guided approach to rapidly and efficiently generate a different variant with recovered affinity. Our case study thus indicates that, while the non-canonical inter-CDR disulfide bond found in rabbit antibodies does not necessarily constitute an obstacle to therapeutic antibody development, combining structure- and ML-guided approaches can provide a fast and efficient way to improve antibody properties and remove potential liabilities.

FlowCam 8400 and FlowCam Cyano Phytoplankton Classification and Viability Staining by Imaging Flow Cytometry.

This chapter provides a protocol for a detailed evaluation of phytoplankton and nuisance cyanobacteria with the FlowCam 8400 and the FlowCam Cyano. The chapter includes (i) detailed description of the quality control of fluorescent mode of the FlowCam, (ii) detailing methods for discriminating nuisance cyanobacteria using the FlowCam Cyano, how to set up libraries and classification routines for commonly used classification reports, and (iii) detailing methods for viability staining to quantify LIVE versus DEAD phytoplankton using the FlowCam 8400.

The effects of heparin, aspirin, and maternal clinical factors on the rate of nonreportable cell-free DNA results: a retrospective cohort study.

BACKGROUND: Technological advances in the analysis of cell-free DNA in maternal serum have allowed expanded prenatal screening possibilities for fetal aneuploidies. The sensitivity and positive predictive value of the assay are partly dependent on the amount of cell-free DNA present in maternal circulation. Thus, it is important to know what fetal and maternal factors influence the level of cell-free DNA in maternal circulation. Maternal heparin use has been associated with an increase in nonreportable cell-free DNA results because of a low fetal fraction in some, but not all, previous studies. In addition, there are likely additional factors that affect cell-free DNA that remain uncharacterized. OBJECTIVE: This study aimed to determine whether heparins, low-dose aspirin, and maternal clinical factors affect the rate of nonreportable cell-free DNA testing results. STUDY DESIGN: A retrospective cohort study was conducted using pregnant people receiving cell-free fetal DNA testing from January 1, 2014, to June 30, 2018. Data were collected on patient demographics, medical comorbidities, medication use, and cell-free DNA test results. Univariate and multivariate analyses were performed to determine which factors were independently associated with the rate of nonreportable results. RESULTS: From an original sample of 1117 pregnant people, 743 met the inclusion criteria. Maternal weight (odds ratio, 1.02), heparin use (odds ratio, 12.06), aspirin use (odds ratio, 4.70), chronic hypertension (odds ratio, 5.26), pregestational diabetes mellitus (odds ratio, 2.46), and autoimmune disease (odds ratio, 3.59) were significantly associated with an increased rate of nonreportable results in the univariate analysis. Moreover, the association was present for maternal weight (odds ratio, 1.02), heparin use (odds ratio, 21.87),and aspirin use (odds ratio, 2.85) in the multivariate analysis. CONCLUSION: The previously seen association between maternal heparin use and an increase in nonreportable cell-free DNA results was confirmed. Furthermore, there seems to be an increase in nonreportable results in pregnant people taking low-dose aspirin. Providers should consider the effect of these medications when counseling patients on prenatal genetic screening options.

Unequal opportunities from the start: Socioeconomic disparities in classroom participation in preschool.

Why do socioeconomic disparities in achievement emerge so early in life? Previous answers to this question have generally focused on the perceived deficits of parents from disadvantaged backgrounds (e.g., insufficient childrearing knowledge). Here, we instead focus on the structure of early childhood education and argue that early schooling contexts provide unequal opportunities for engagement to children of higher versus lower socioeconomic status (SES). As engagement is a longitudinal predictor of achievement, early SES disparities in engagement could serve to maintain or even exacerbate SES disparities in achievement. In Study 1 (1,236 observations; N = 98 children), we investigated preschool students' behavioral engagement during whole-class discussions-a core aspect of early childhood education. Low-SES children showed significantly lower engagement than their peers. Consistent with the claim of unequal opportunities for engagement, these differences were not accounted for by SES differences in language proficiency. As students' engagement in school is influenced by their peers' attitudes toward them, we also examined peer perceptions (Study 2, N = 94, and a meta-analysis, k = 2 studies). We found that preschoolers who show more engagement relative to others during whole-class discussions are perceived as possessing more positive qualities (e.g., intelligence). Given that higher-SES students are afforded more opportunities for engagement (see Study 1), they may be the ones benefiting from these positive peer perceptions as well, which might further boost their engagement. Our results suggest that aspects of early childhood education should be redesigned to foster engagement among all students, regardless of their SES. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Personal harm from the Covid-19 pandemic predicts advocacy for equality.

The Covid-19 pandemic has laid bare the vast amount of economic inequality in the U.S. Yet, has it influenced Americans' attitudes and behaviors toward equality? With a three-wave longitudinal survey, the current research provides evidence that experiencing personal harm (e.g., contracting Covid-19, losing jobs, or psychological distress) from the pandemic predicts an increase in people's attitudinal and behavioral advocacy for equality. Specifically, we find that experiencing greater personal harm in the early stages of the pandemic (i.e., May 2020) is associated with increased advocacy for equality one year later (i.e., May 2021; e.g., contacting a public official to express support for reducing inequality). Furthermore, we find that this increase in advocacy for equality is explained, in part, by people's greater endorsement of the external factors (e.g., bad luck, discrimination, etc.) that contribute to inequality. Our work provides evidence that the extent to which people experience harm from the Covid-19 pandemic predicts both their increased understanding of external sources of inequality, as well as their efforts to combat this inequality (e.g., by advocating for policies that combat structural contributors to inequality).

Comprehensive UHPLC- and CE-based methods for engineered Cas9 characterization.

CRISPR (clustered regularly interspaced short palindromic repeats)-associated proteins (Cas) are powerful gene-editing tools used in therapeutic applications. Efforts to minimize off-target cleavage by CRISPR-Cas9 have motivated the development of engineered Cas9 variants. The wild-type (WT) Streptococcus pyogenes (SpCas9) has been engineered into a high-fidelity Cas9 (SpyFi Cas9) that shows promising results in providing high on-target activity (targeting efficiency) while reducing off-target editing (unwanted mutations). This work describes for the first time the development of ultra-high-performance liquid chromatography (UHPLC) and capillary electrophoresis (CE)-based methods for a full characterization of different engineered Cas9 variants, including determination of purity, size variants, isoelectric points (pI), post-translational modifications (PTMs), and functional activities. The purity and size variant characterization were first determined by CE-sodium dodecyl sulfate (SDS). An in vitro DNA cleavage assay using an automated electrophoresis tool was employed to investigate the functional activity of ribonucleoprotein (RNP) complexes derived from Cas9 variants. The pIs of the engineered Cas9 proteins were determined by imaged capillary isoelectric focusing (icIEF), while intact mass measurements were performed by reversed-phase (RP)-UHPLC coupled with high-resolution mass spectrometry (HRMS). A peptide mapping assay based on LC-UV-MS/MS using endoproteinase Lys-C under non-reducing conditions was developed to confirm amino acid sequences, allowing differentiation of SpyFi Cas9 from WT SpCas9. The potential of using a low-resolution MS detector, especially for a GMP environment, as a low-cost and simple method to identify SpyFi Cas9 is discussed.

Decision-Making Processes of Caregivers Choosing an Educational Setting for Children Who Are Deaf or Hard of Hearing.

Deciding on an educational setting for children who are deaf or hard of hearing (DHH) is a complex process that is not well understood. In the present study, the researchers' objective was to understand the factors caregivers consider when choosing a school for their child. Six caregivers of children who were DHH participated in semistructured interviews, which were coded into three themes (Child-Centered, Familial, School) and five subthemes (Inclusion, Additional Needs and Well-Being, Complex Process, Information Input and Flow, School Systems and Personnel). An unexpected theme (On Reflection) and three additional subthemes (Caregiver Perceptions of Education, School Character, No Regrets) were also identified. A highlighted finding is that when choosing an educational setting, caregivers of children who are DHH use decision-making processes that are complex and multifaceted. Practical implications for professionals supporting caregivers through decision-making processes are outlined, and applications for practice are suggested.

Is diversity enough? Cross-race and cross-class interactions in college occur less often than expected, but benefit members of lower status groups when they occur.

More than ever before, institutions of higher education are seeking to increase the racial and social class diversity of their student bodies. Given these efforts, the present research asks two broad questions. First, how frequently do intergroup interactions occur across the lines of race and social class, and to what extent do these interactions reflect the diversity of a setting? Second, when cross-race and cross-class interactions occur, how do individuals experience them and what consequences do they have for their outcomes in these settings? Leveraging a longitudinal design and daily diary methods, we conducted the first large study (Ninteractions = 11,460) which tracks the frequency, experience, and consequences of meaningful cross-race and cross-class interactions. We found that students reported far fewer cross-race and cross-class interactions than would occur at chance given the racial and social class diversity of their student bodies. Furthermore, students experienced less satisfaction and perspective-taking in cross-race and cross-class interactions compared to same-race and same-class interactions, respectively. Nevertheless, these cross-group interactions predicted better academic performance for underrepresented racial minority students and students from working and lower class backgrounds. They did so, in part, by increasing students' feelings of inclusion (i.e., increased belonging and reduced social identity threat). Together, these findings suggest that the mere presence of diversity is not enough to foster meaningful intergroup interactions. Furthermore, fostering intergroup interactions may be one important pathway toward reducing racial and social class disparities. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Temperature-Dependent Constrained Diffusion of Micro-Confined Alkylimidazolium Chloride Ionic Liquids.

Alkylimidazolium chloride ionic liquids (ILs) have many uses in a variety of separation systems, including micro-confined separation systems. To understand the separation mechanism in these systems, the diffusion properties of analytes in ILs under relevant operating conditions, including micro-confinement dimension and temperature, should be known. For example, separation efficiencies for various IL-based microextraction techniques are dependent on the sample volume and temperature. Temperature-dependent (20-100 °C) fluorescence recovery after photobleaching (FRAP) was utilized to determine the diffusion properties of a zwitterionic, hydrophilic dye, ATTO 647, in alkylimidazolium chloride ILs in micro-confined geometries. These micro-confined geometries were generated by sandwiching the IL between glass substrates that were separated by ∼1 to 100 µm. From the measured temperature-dependent FRAP data, we note alkyl chain length-, thickness-, and temperature-dependent diffusion coefficients, with values ranging from 0.021 to 46 µm2/s. Deviations from Brownian diffusion are observed at lower temperatures and increasingly less so at elevated temperatures; the differences are attributed to alterations in intermolecular interactions that reduce temperature-dependent nanoscale structural heterogeneities. The temperature- and thickness-dependent data provide a useful foundation for efficient design of micro-confined IL separation systems.