Parents' color-blind racial ideology and implicit racial attitudes predict children's race-based sympathy.

We examined the relation of White parents' color-blind racial attitudes (a global composite score and its subscales) and their implicit racial attitudes to their young children's race-based sympathy toward Black and White victims. One hundred and nighty non-Hispanic White children (54% boys, Mage = 7.13 years, SD = 0.92) reported their sympathy in response to short films depicting bullying toward White or Black children. Their primary caregivers' (mostly mothers') color-blind racial ideology (CBRI) was assessed through a questionnaire (reflecting global color blindness, as well as denial of institutional racism, White privilege, and blatant racial issues), and their implicit racial attitudes were assessed with a computerized test. Children's sympathy toward Black victims and their equitable sympathy (difference score toward Black vs. White victims) was predicted by parents' color blindness, implicit racial attitudes, and their interaction. Results indicated several interaction effects, such that parents' denial of blatant racial attitudes and global CBRI were negatively related to children's sympathy toward Black victims and equitable sympathy toward Black versus White victims, only when the parents held implicit racial attitudes that favored White people. In addition, parents' denial of White privilege was negatively related to children's sympathy toward Black victims. The findings are discussed in terms of potential ways to shape children's race-based sympathy and compassion, particularly with an eye toward ways White parents might socialize sympathy toward historically marginalized youth. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

White children's empathy-related responding and prosocial behavior toward White and Black children.

Relations among White (non-Latinx) children's empathy-related responding, prosocial behaviors, and racial attitudes toward White and Black peers were examined. In 2017, 190 (54% boys) White 5- to 9-year-old children (M = 7.09 years, SD = 0.94) watched a series of videos that depicted social rejection of either a White or Black child. Empathy-related responses, prosocial behaviors, and racial attitudes were measured using multiple methods. Results showed that younger children showed less facial concern toward Black than White peers and greater increases with age in concern and prosocial behaviors (sharing a desirable prize) for Black, compared to White, targets. Children's facial anger increased with age for White but not Black targets. The findings can extend our understanding children's anti-racism development.

Cross-Sectional Analysis of Burnout, Secondary Traumatic Stress, and Compassion Satisfaction Among Emergency Nurses in Southern California Working Through the COVID-19 Pandemic.

OBJECTIVE: The purpose of this study was to assess burnout, secondary traumatic stress, and compassion satisfaction scores in emergency nurses after working through the COVID-19 pandemic using the Professional Quality of Life Scale version 5 and compare those scores with similar studies conducted before the pandemic. METHODS: A cross-sectional analysis of a descriptive survey including the Professional Quality of Life Scale version 5 questionnaire was sent to nurses working in the emergency department before 2021 from urban, adult, and pediatric receiving hospitals in Southern California. Results were analyzed to provide insight into the effect of the COVID-19 pandemic on the levels of burnout, secondary traumatic stress, and compassion satisfaction compared with prepandemic studies found in the literature using the same Professional Quality of Life Scale version 5 instrument. RESULTS: Mean subcategory scores were in the moderate range for burnout (25.6), secondary traumatic stress (24.5), and compassion satisfaction (38.7). Burnout scores for midshift nurses were found to be significantly higher than day shift nurses (mean difference 5, P = .02) as were secondary traumatic stress scores (mean difference 4.6, P = .007). In addition, compassion satisfaction subcategory scores in nurses with 1 child living at home were significantly higher than those with 2 (mean difference 6.7, P = .02). DISCUSSION: The unnormalized mean findings were similar to prepandemic studies conducted using the same Professional Quality of Life Scale version 5 instrument indicating nurses are at risk of compassion fatigue. In addition, the scores from midshift nurses reflect increased burnout and secondary traumatic stress whereas nurses with 2 children had lower compassion satisfaction. This implies the need for leadership to proactively seek interventions to support nurses on each shift.

Longitudinal relations among household chaos, SES, and effortful control in the prediction of language skills in early childhood.

Previous research has shown that home environment plays an important role in children's early language skills. Yet, few researchers have examined the unique role of family-level factors (socioeconomic status [SES], household chaos) on children's learning or focused on the longitudinal processes that might explain their relations to children's early language skills. The goal of this study was to investigate the longitudinal relations from family SES, household chaos, and children's effortful control (EC) to children's language skills during early childhood, controlling for stability of the constructs. At Time (T1 i.e., 30 months), mothers reported family SES and children's vocabulary, and their own linguistic input was assessed during a free-play session with their child. At T1, T2 (i.e., 42 months), and T3 (i.e., 54 months), household chaos was reported by mothers, and children's EC was rated by mothers and nonparental caregivers and observed during a gift-delay task. At T3, children's expressive and receptive language skills were measured with a standard assessment. Path analyses indicated that higher SES predicted higher levels of EC at T2 and language skills at T3, and greater levels of household chaos at T2 predicted poorer EC and language skills a year later, even when controlling for stability of the constructs. Results indicated that T2 EC partially mediated the relations between SES and T3 language skills. Findings from this study can be used to identify key factors for early learning and perhaps inform programs designed to support families and young children. (PsycINFO Database Record (c) 2020 APA, all rights reserved).

Lipid order and charge protect killer T cells from accidental death.

Killer T cells (cytotoxic T lymphocytes, CTLs) maintain immune homoeostasis by eliminating virus-infected and cancerous cells. CTLs achieve this by forming an immunological synapse with their targets and secreting a pore-forming protein (perforin) and pro-apoptotic serine proteases (granzymes) into the synaptic cleft. Although the CTL and the target cell are both exposed to perforin within the synapse, only the target cell membrane is disrupted, while the CTL is invariably spared. How CTLs escape unscathed remains a mystery. Here, we report that CTLs achieve this via two protective properties of their plasma membrane within the synapse: high lipid order repels perforin and, in addition, exposed phosphatidylserine sequesters and inactivates perforin. The resulting resistance of CTLs to perforin explains their ability to kill target cells in rapid succession and to survive these encounters. Furthermore, these mechanisms imply an unsuspected role for plasma membrane organization in protecting cells from immune attack.

Bi-Allelic Mutations in STXBP2 Reveal a Complementary Role for STXBP1 in Cytotoxic Lymphocyte Killing.

The ability of cytotoxic lymphocytes (CL) to eliminate virus-infected or cancerous target cells through the granule exocytosis death pathway is critical to immune homeostasis. Congenital loss of CL function due to bi-allelic mutations in PRF1, UNC13D, STX11, or STXBP2 leads to a potentially fatal immune dysregulation, familial haemophagocytic lymphohistiocytosis (FHL). This occurs due to the failure of CLs to release functional pore-forming protein perforin and, therefore, inability to kill the target cell. Bi-allelic mutations in partner proteins STXBP2 or STX11 impair CL cytotoxicity due to failed docking/fusion of cytotoxic secretory granules with the plasma membrane. One unique feature of STXBP2- and STX11-deficient patient CLs is that their short-term in vitro treatment with a low concentration of IL-2 partially or completely restores natural killer (NK) cell degranulation and cytotoxicity, suggesting the existence of a secondary, yet unknown, pathway for secretory granule exocytosis. In the current report, we studied NK and T-cell function in an individual with late presentation of FHL due to hypomorphic bi-allelic mutations in STXBP2. Intriguingly, in addition to the expected alterations in the STXBP2 and STX11 proteins, we also observed a concomitant significant reduction in the expression of homologous STXBP1 protein and its partner STX1, which had never been implicated in CL function. Further analysis of human NK and T cells demonstrated a functional role for the STXBP1/STX1 axis in NK and CD8+ T-cell cytotoxicity, where it appears to be responsible for as much as 50% of their cytotoxic activity. This discovery suggests a unique and previously unappreciated interplay between STXBP/Munc proteins regulating the same essential granule exocytosis pathway.

Failed CTL/NK cell killing and cytokine hypersecretion are directly linked through prolonged synapse time.

Failure of cytotoxic T lymphocytes (CTLs) or natural killer (NK) cells to kill target cells by perforin (Prf)/granzyme (Gzm)-induced apoptosis causes severe immune dysregulation. In familial hemophagocytic lymphohistiocytosis, Prf-deficient infants suffer a fatal "cytokine storm" resulting from macrophage overactivation, but the link to failed target cell death is not understood. We show that prolonged target cell survival greatly amplifies the quanta of inflammatory cytokines secreted by CTLs/NK cells and that interferon-γ (IFN-γ) directly invokes the activation and secondary overproduction of proinflammatory IL-6 from naive macrophages. Furthermore, using live cell microscopy to visualize hundreds of synapses formed between wild-type, Prf-null, or GzmA/B-null CTLs/NK cells and their targets in real time, we show that hypersecretion of IL-2, TNF, IFN-γ, and various chemokines is linked to failed disengagement of Prf- or Gzm-deficient lymphocytes from their targets, with mean synapse time increased fivefold, from ∼8 to >40 min. Surprisingly, the signal for detachment arose from the dying target cell and was caspase dependent, as delaying target cell death with various forms of caspase blockade also prevented their disengagement from fully competent CTLs/NK cells and caused cytokine hypersecretion. Our findings provide the cellular mechanism through which failed killing by lymphocytes causes systemic inflammation involving recruitment and activation of myeloid cells.

The RabGAP TBC1D1 plays a central role in exercise-regulated glucose metabolism in skeletal muscle.

Insulin and exercise stimulate glucose uptake into skeletal muscle via different pathways. Both stimuli converge on the translocation of the glucose transporter GLUT4 from intracellular vesicles to the cell surface. Two Rab guanosine triphosphatases-activating proteins (GAPs) have been implicated in this process: AS160 for insulin stimulation and its homolog, TBC1D1, are suggested to regulate exercise-mediated glucose uptake into muscle. TBC1D1 has also been implicated in obesity in humans and mice. We investigated the role of TBC1D1 in glucose metabolism by generating TBC1D1(-/-) mice and analyzing body weight, insulin action, and exercise. TBC1D1(-/-) mice showed normal glucose and insulin tolerance, with no difference in body weight compared with wild-type littermates. GLUT4 protein levels were reduced by ∼40% in white TBC1D1(-/-) muscle, and TBC1D1(-/-) mice showed impaired exercise endurance together with impaired exercise-mediated 2-deoxyglucose uptake into white but not red muscles. These findings indicate that the RabGAP TBC1D1 plays a key role in regulating GLUT4 protein levels and in exercise-mediated glucose uptake in nonoxidative muscle fibers.

Exploration of a series of 5-arylidene-2-thioxoimidazolidin-4-ones as inhibitors of the cytolytic protein perforin.

A series of novel 5-arylidene-2-thioxoimidazolidin-4-ones were investigated as inhibitors of the lymphocyte-expressed pore-forming protein perforin. Structure-activity relationships were explored through variation of an isoindolinone or 3,4-dihydroisoquinolinone subunit on a fixed 2-thioxoimidazolidin-4-one/thiophene core. The ability of the resulting compounds to inhibit the lytic activity of both isolated perforin protein and perforin delivered in situ by natural killer cells was determined. A number of compounds showed excellent activity at concentrations that were nontoxic to the killer cells, and several were a significant improvement on previous classes of inhibitors, being substantially more potent and soluble. Representative examples showed rapid and reversible binding to immobilized mouse perforin at low concentrations (≤2.5 µM) by surface plasmon resonance and prevented formation of perforin pores in target cells despite effective target cell engagement, as determined by calcium influx studies. Mouse PK studies of two analogues showed T1/2 values of 1.1-1.2 h (dose of 5 mg/kg i.v.) and MTDs of 60-80 mg/kg (i.p.).

Defining the interaction of perforin with calcium and the phospholipid membrane.

Following its secretion from cytotoxic lymphocytes into the immune synapse, perforin binds to target cell membranes through its Ca(2+)-dependent C2 domain. Membrane-bound perforin then forms pores that allow passage of pro-apoptopic granzymes into the target cell. In the present study, structural and biochemical studies reveal that Ca(2+) binding triggers a conformational change in the C2 domain that permits four key hydrophobic residues to interact with the plasma membrane. However, in contrast with previous suggestions, these movements and membrane binding do not trigger irreversible conformational changes in the pore-forming MACPF (membrane attack complex/perforin-like) domain, indicating that subsequent monomer-monomer interactions at the membrane surface are required for perforin pore formation.

Comparative studies of Munc18c and Munc18-1 reveal conserved and divergent mechanisms of Sec1/Munc18 proteins.

Sec1/Munc18 (SM) family proteins are essential for every vesicle fusion pathway. The best-characterized SM protein is the synaptic factor Munc18-1, but it remains unclear whether its functions represent conserved mechanisms of SM proteins or specialized activities in neurotransmitter release. To address this question, we dissected Munc18c, a functionally distinct SM protein involved in nonsynaptic exocytic pathways. We discovered that Munc18c binds to the trans-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex and strongly accelerates the fusion rate. Further analysis suggests that Munc18c recognizes both vesicle-rooted SNARE and target membrane-associated SNAREs, and promotes trans-SNARE zippering at the postdocking stage of the fusion reaction. The stimulation of fusion by Munc18c is specific to its cognate SNARE isoforms. Because Munc18-1 regulates fusion in a similar manner, we conclude that one conserved function of SM proteins is to bind their cognate trans-SNARE complexes and accelerate fusion kinetics. Munc18c also binds syntaxin-4 monomer but does not block target membrane-associated SNARE assembly, in agreement with our observation that six- to eightfold increases in Munc18c expression do not inhibit insulin-stimulated glucose uptake in adipocytes. Thus, the inhibitory "closed" syntaxin binding mode demonstrated for Munc18-1 is not conserved in Munc18c. Unexpectedly, we found that Munc18c recognizes the N-terminal region of the vesicle-rooted SNARE, whereas Munc18-1 requires the C-terminal sequences, suggesting that the architecture of the SNARE/SM complex likely differs across fusion pathways. Together, these comparative studies of two distinct SM proteins reveal conserved as well as divergent mechanisms of SM family proteins in intracellular vesicle fusion.

Rapid and unidirectional perforin pore delivery at the cytotoxic immune synapse.

The effective engagement of cytotoxic lymphocytes (CLs) with their target cells is essential for the removal of virus-infected and malignant cells from the body. The spatiotemporal properties that define CL engagement and killing of target cells remain largely uncharacterized due to a lack of biological reporters. We have used a novel live cell microscopy technique to visualize the engagement of primary human and mouse CL with their targets and the subsequent delivery of the lethal hit. Extensive quantitative real-time analysis of individual effector-target cell conjugates demonstrated that a single effector calcium flux event was sufficient for the degranulation of human CLs, resulting in the breach of the target cell membrane by perforin within 65-100 s. In contrast, mouse CLs demonstrated distinct calcium signaling profiles leading to degranulation: whereas mouse NKs required a single calcium flux event, CD8(+) T cells typically required several calcium flux events before perforin delivery. Irrespective of their signaling profile, every target cell that was damaged by perforin died by apoptosis. To our knowledge, we demonstrate for the first time that perforin pore delivery is unidirectional, occurring exclusively on the target cell membrane, but sparing the killer cell. Despite this, the CTL membrane was not intrinsically perforin resistant, as intact CTLs presented as targets to effector CTLs were capable of being killed by perforin-dependent mechanisms. Our results highlight the remarkable efficiency and specificity of perforin pore delivery by CLs.

Perforin forms transient pores on the target cell plasma membrane to facilitate rapid access of granzymes during killer cell attack.

Cytotoxic lymphocytes serve a key role in immune homeostasis by eliminating virus-infected and transformed target cells through the perforin-dependent delivery of proapoptotic granzymes. However, the mechanism of granzyme entry into cells remains unresolved. Using biochemical approaches combined with time-lapse microscopy of human primary cytotoxic lymphocytes engaging their respective targets, we defined the time course of perforin pore formation in the context of the physiological immune synapse. We show that, on recognition of targets, calcium influx into the lymphocyte led to perforin exocytosis and target cell permeabilization in as little as 30 seconds. Within the synaptic cleft, target cell permeabilization by perforin resulted in the rapid diffusion of extracellular milieu-derived granzymes. Repair of these pores was initiated within 20 seconds and was completed within 80 seconds, thus limiting granzyme diffusion. Remarkably, even such a short time frame was sufficient for the delivery of lethal amounts of granzymes into the target cell. Rapid initiation of apoptosis was evident from caspase-dependent target cell rounding within 2 minutes of perforin permeabilization. This study defines the final sequence of events controlling cytotoxic lymphocyte immune defense, in which perforin pores assemble on the target cell plasma membrane, ensuring efficient delivery of lethal granzymes.

RalA GTPase tethers insulin granules to L- and R-type calcium channels through binding α2 δ-1 subunit.

RalA GTPase has been implicated in the regulated delivery of exocytotic vesicles to the plasma membrane (PM) in mammalian cells. We had reported that RalA regulates biphasic insulin secretion, which we have now determined to be contributed by RalA direct interaction with voltage-gated calcium (Cav ) channels. RalA knockdown (KD) in INS-1 cells and primary rat ß-cells resulted in a reduction in Ca(2+) currents arising specifically from L-(Cav 1.2 and Cav 1.3) and R-type (Cav 2.3) Ca(2+) channels. Restoration of RalA expression in RalA KD cells rescued these defects in Ca(2+) currents. RalA co-immunoprecipitated with the Cav α2 δ-1 auxiliary subunit known to bind the three Cav s. Moreover, the functional molecular interactions between Cav α2 δ-1 and RalA on the PM shown by total internal reflection fluorescent microscopy/FRET analysis could be induced by glucose stimulation. KD of RalA inhibited trafficking of α2 δ-1 to insulin granules without affecting the localization of the other Cav subunits. Furthermore, we confirmed that RalA and α2 δ-1 functionally interact since RalA KD-induced inhibition of Cav currents could not be recovered by RalA when α2 δ-1 was simultaneously knocked down. These data provide a mechanism for RalA function in insulin secretion, whereby RalA binds α2 δ-1 on insulin granules to tether these granules to PM Ca(2+) channels. This acts as a chaperoning step prior to and in preparation for sequential assembly of exocyst and excitosome complexes that mediate biphasic insulin secretion.

Deciphering the syntax of cytotoxic lymphocyte degranulation.

In the killer lymphocyte, the targeted delivery of perforin- and granzyme-containing cytotoxic granules to the immunological synapse is crucial for the eradication of pathogen-infected or transformed targets. This process is achieved through a tightly controlled and highly efficient granule exocytosis pathway. Mutations in the granule trafficking proteins Munc13-4, syntaxin 11, Munc18-2 or Rab27 leads to a fatal lapse of immune surveillance and can be manifested as haemophagocytic lymphohistiocytosis in humans. Elucidation of the role of these proteins in exocytic trafficking is pivotal for our understanding of their role in health and disease. In this issue of the European Journal of Immunology, D'Orlando et al. [Eur. J. Immunol. 2013. 43: 194-208] make an important step in this direction, as they generate and characterise syntaxin 11 deficient mice. Herein, we discuss the role of syntaxin-11 in soluble NSF (N-ethylmaleimide sensitive fusion) attachment protein receptors complex formation leading to cytotoxic lymphocyte degranulation and its importance in maintaining immune homeostasis.

Protecting a serial killer: pathways for perforin trafficking and self-defence ensure sequential target cell death.

Considerable progress has been made in understanding how cytotoxic lymphocytes use the highly toxic pore-forming protein perforin to eliminate dangerous cells, while remaining refractory to lysis. At least two mechanisms jointly preserve the killer cell: the C-terminal residues of perforin dictate its rapid export from the endoplasmic reticulum (ER), whose milieu otherwise favours pore formation; perforin is then stored in secretory granules whose acidity prevent its oligomerisation. Following exocytosis, perforin delivers the proapoptotic protease, granzyme B, into the target cell by disrupting its plasma membrane. Although the precise mechanism of perforin/granzyme synergy remains controversial, the recently defined crystal structure of the perforin monomer and cryo-electron microscopy (EM) of the entire pore suggest that passive transmembrane granzyme diffusion is the dominant proapoptotic mechanism.

Fatal immune dysregulation due to a gain of glycosylation mutation in lymphocyte perforin.

Mutations in the perforin gene (PRF1) are a common cause of the fatal immune dysregulation disorder, familial hemophagocytic lymphohistiocytosis (type 2 FHL, FHL2). Here we report a female infant born with biallelic PRF1 mutations: a novel substitution, D49N, and a previously identified in-frame deletion, K285del. We assessed the effects of each mutation on the cytotoxicity of human NK cells in which the expression of endogenous perforin was ablated with miR30-based short hairpin (sh) RNAs. Both mutations were detrimental for function, thereby explaining the clinically severe presentation and rapidly fatal outcome. We demonstrate that D49N exerts its deleterious effect by generating an additional (third) N-linked glycosylation site, resulting in protein misfolding and degradation in the killer cell. Our data provide a rationale for treating some cases of type 2 familial hemophagocytic lymphohistiocytosis, based on the pharmacologic inhibition or modification of glycosylation.

Protection from endogenous perforin: glycans and the C terminus regulate exocytic trafficking in cytotoxic lymphocytes.

Cytotoxic lymphocyte-mediated apoptosis is dependent on the delivery of perforin to secretory granules and its ability to form calcium-dependent pores in the target cell after granule exocytosis. It is unclear how cytotoxic lymphocytes synthesize and store perforin without incurring damage or death. We discovered that the extreme C terminus of perforin was essential for rapid trafficking from the endoplasmic reticulum to the Golgi compartment. Substitution of the C-terminal tryptophan residue resulted in retention of perforin in the ER followed by calcium-dependent toxic activity that eliminated host cells. We also found that N-linked glycosylation of perforin was critical for transport from the Golgi to secretory granules. Overall, an intact C terminus and N-linked glycosylation provide accurate and efficient export of perforin from the endoplasmic reticulum to the secretory granules and are critical for cytotoxic lymphocyte survival.

Religious commitment, adult attachment, and marital adjustment in newly married couples.

Existing literature on the role of religiosity in marital functioning is often difficult to interpret due to the frequent use of convenience samples, statistical approaches inadequate for interdependent dyadic data, and the lack of a theoretical framework. The current study examined the effects of religious commitment and insecure attachment on marital adjustment. Newly married couples who did not have children (N = 92 couples, 184 individuals) completed measures of religious commitment, adult attachment, and marital functioning. There was a small positive association between religious commitment and marital adjustment. Religious commitment buffered the negative association between attachment avoidance and marital adjustment, but exacerbated the negative association between attachment anxiety and marital adjustment.