The Chalcidoidea bush of life: evolutionary history of a massive radiation of minute wasps.

Chalcidoidea are mostly parasitoid wasps that include as many as 500 000 estimated species. Capturing phylogenetic signal from such a massive radiation can be daunting. Chalcidoidea is an excellent example of a hyperdiverse group that has remained recalcitrant to phylogenetic resolution. We combined 1007 exons obtained with Anchored Hybrid Enrichment with 1048 ultra-conserved elements (UCEs) for 433 taxa including all extant families, >95% of all subfamilies, and 356 genera chosen to represent the vast diversity of the superfamily. Going back and forth between the molecular results and our collective knowledge of morphology and biology, we detected bias in the analyses that was driven by the saturation of nucleotide data. Our final results are based on a concatenated analysis of the least saturated exons and UCE datasets (2054 loci, 284 106 sites). Our analyses support an expected sister relationship with Mymarommatoidea. Seven previously recognized families were not monophyletic, so support for a new classification is discussed. Natural history in some cases would appear to be more informative than morphology, as illustrated by the elucidation of a clade of plant gall associates and a clade of taxa with planidial first-instar larvae. The phylogeny suggests a transition from smaller soft-bodied wasps to larger and more heavily sclerotized wasps, with egg parasitism as potentially ancestral for the entire superfamily. Deep divergences in Chalcidoidea coincide with an increase in insect families in the fossil record, and an early shift to phytophagy corresponds with the beginning of the "Angiosperm Terrestrial Revolution". Our dating analyses suggest a middle Jurassic origin of 174 Ma (167.3-180.5 Ma) and a crown age of 162.2 Ma (153.9-169.8 Ma) for Chalcidoidea. During the Cretaceous, Chalcidoidea may have undergone a rapid radiation in southern Gondwana with subsequent dispersals to the Northern Hemisphere. This scenario is discussed with regard to knowledge about the host taxa of chalcid wasps, their fossil record and Earth's palaeogeographic history.

1,8-Dihydroxy Naphthalene-A New Building Block for the Self-Assembly with Boronic Acids and 4,4'-Bipyridine to Stable Host-Guest Complexes with Aromatic Hydrocarbons.

The new Lewis acid-base adducts of general formula X(nad)B←NC5H4-C5H4N→B(nad)X [nad = 1,8-O2C10H6, X = C6H5 (2c), 3,4,5-F3-C6H2 (2d)] were synthesized in high yields via reactions of 1,8-dihydroxy naphthalene [nadH2] and 4,4'-bipyridine with the aryl boronic acids C6H5B(OH)2 and 3,4,5-F3-C6H2B(OH)2, respectively, and structurally characterized by multi-nuclear NMR spectroscopy and SCXRD. Self-assembled H-shaped Lewis acid-base adduct 2d proved to be effective in forming thermally stable host-guest complexes, 2d × solvent, with aromatic hydrocarbon solvents such as benzene, toluene, mesitylene, aniline, and m-, p-, and o-xylene. Crystallographic analysis of these solvent adducts revealed host-guest interactions to primarily occur via π···π contacts between the 4,4'-bipyridyl linker and the aromatic solvents, resulting in the formation of 1:1 and 1:2 host-guest complexes. Thermogravimetric analysis of the isolated complexes 2d × solvent revealed their high thermal stability with peak temperatures associated with the loss of solvent ranging from 122 to 147 °C. 2d, when self-assembled in an equimolar mixture of m-, p-, and o-xylene (1:1:1), preferentially binds to o-xylene. Collectively, these results demonstrate the ability of 1,8-dihydroxy naphthalene to serve as an effective building block in the selective self-assembly to supramolecular aggregates through dative covalent N→B bonds.

Resilient Parents Resilient Communities: A Pilot Study Trialing the Bounce Back and Thrive! Resilience-Training Program With Military Families.

INTRODUCTION: The resilience of Canadian military families (CMFs) - the main support of the Canadian Armed Forces service members (SMs) - is imperative. The Canadian Armed Forces aims to ensure that SMs and their families are resilient and SMs ready to respond when called upon for combat, peacekeeping or pandemic/disaster-response. Family concerns, however, can realistically distract SMs from the mission, potentially compromising themselves, their unit and the mission. Resilience-training programs such as Bounce Back and Thrive! (BBT) can help families manage the realities of military life. OBJECTIVE: This pilot study aimed to evaluate suitability of BBT implementation by Military Family Resource Centers (MFRCs), including whether BBT: (1) fosters resilience-building among parents, (2) facilitates CMF resilience-building, (3) can be contextualized for CMFs, and (4) supports MFRCs in cultivating a culture of resilience. METHODS: An exploratory qualitative design was used. BBT was offered to parents face-to-face. Participants completed focus groups after the first 6 sessions, final 4 sessions, and one-year post-intervention. Data was thematically analyzed. RESULTS: Nine military parents participated. Four major themes resulted: (1) military parent resilience-building, (2) CMF resilience-building, (3) BBT program feedback and contextualization, and (4) MFRCs as community resilience hubs. DISCUSSION: BBT enabled parents to gain a new perspective on resilience, engage in dialogue and intentionally role model resilience skills. Military-specific BBT contextualization and online-delivery formats would increase suitability and access for CMFs. Access to resilience programs delivered through MFRCs would support CMFs. Further research is warranted.

Updated systematic review and meta-analysis of studies examining the relationship between reported racism and health and well-being for children and youth: a protocol.

INTRODUCTION: Racism is a critical determinant of health and health inequities for children and youth. This protocol aims to update the first systematic review conducted by Priest et al (2013), including a meta-analysis of findings. Based on previous empirical data, it is anticipated that child and youth health will be negatively impacted by racism. Findings from this review will provide updated evidence of effect sizes across outcomes and identify moderators and mediators of relationships between racism and health. METHODS AND ANALYSIS: This systematic review and meta-analysis will include studies that examine associations between experiences of racism and racial discrimination with health outcomes of children and youth aged 0-24 years. Exposure measures include self-reported or proxy reported systemic, interpersonal and intrapersonal racism. Outcome measures include general health and well-being, physical health, mental health, biological markers, healthcare utilisation and health behaviours. A comprehensive search of studies from the earliest time available to October 2020 will be conducted. A random effects meta-analysis will examine the average effect of racism on a range of health outcomes. Study-level moderation will test the difference in effect sizes with regard to various sample and exposure characteristics. This review has been registered with the International Prospective Register of Systematic Reviews. ETHICS AND DISSEMINATION: This review will provide evidence for future research within the field and help to support policy and practice development. Results will be widely disseminated to both academic and non-academic audiences through peer-review publications, community summaries and presentations to research, policy, practice and community audiences. PROSPERO REGISTRATION NUMBER: CRD42020184055.

Enhancing Resilience in Canadian Military Families and Communities: A Qualitative Analysis of the Reaching In Reaching Out and Bounce Back and Thrive! Resiliency Skills Training Programs.

Introduction: A new vision of resilience and well-being for Canadian military service members (SMs), Veterans and their families has been championed by the Canadian Armed Forces (CAF) and Veterans Affairs Canada (VAC). Operationalizing this vision, which aims to support those who serve/have served and their families as they navigate life during and post-service, requires the support of service providers (SPs). Training SPs to deliver complementary resilience-training programs Reaching In… Reaching Out (RIRO; for adults working with parents of young children) and Bounce Back and Thrive! (BBT; for parents of children aged 0-8 years of age) may support this vision. Objective: To assess the appropriateness of RIRO/BBT trainer training for SPs, and RIRO and BBT resilience-training for military populations and families. Methods: This qualitative descriptive study involved the delivery of RIRO/BBT trainer training to SPs (n = 20), followed by focus groups (n = 6) with SPs and organisational leaders (n = 4). Focus groups were recorded, and data were transcribed and thematically-analysed. Results: Several themes emerged: (1) RIRO/BBT trainer training enabled SPs to model resilience and deliver the resilience-training programs, (2) training was appropriate and adaptable for the CAF and SMs/CMFs, and (3) training could support the development of resilient communities. Discussion: RIRO/BBT trainer training and RIRO and BBT resilience-training programs use a holistic, integrated, experiential, and community approach to resilience-building and align with CAF and VAC initiatives. Once contextualised, such programs could support resilience-building in the military context.

Read between the lineata: A revision of the tattooed wasps, Zagrammosoma Ashmead (Hymenoptera: Eulophidae), with descriptions of eleven new species.

The genus Zagrammosoma Ashmead (Hymenoptera: Eulophidae) is revised. Twenty-six species are recognized, of which 23 have been molecularly verified in a phylogenetic context using 28S, ITS2, and COI. Zagrammosoma is recovered as monophyletic, worldwide in distribution, and morphologically distinct from Cirrospilus Westwood. Zagrammosoma interlineatum Girault stat. rev. is elevated from synonymy. The following synonymies are proposed: Zagrammosoma mirum Girault under Zagrammosoma flavolineatum Crawford n. syn., Zagrammosoma dulanense Cao Zhu under Zagrammosoma talitzkii Boucek n. syn. The following species are described as new: Zagrammosoma calvini Perry n. sp. (Argentina, Chile), Zagrammosoma deliae Perry n. sp. (Peru), Zagrammosoma fisheri Perry n. sp. (USA), Zagrammosoma galapagoense Perry n. sp. (Ecuador: Galapagos Islands), Zagrammosoma headricki Perry n. sp. (Mexico, USA), Zagrammosoma metallicum Perry n. sp. (USA), Zagrammosoma occidentale Perry n. sp. (Mexico, USA), Zagrammosoma triangulum Perry n. sp. (USA), Zagrammosoma trifurcatum Perry n. sp. (Belize, USA), Zagrammosoma villosum Perry n. sp. (Nearctic, Neotropical), Zagrammosoma yanegai Perry n. sp. (Thailand). The following new combination is proposed: Cirrospilus variegatus (Masi) n. comb, from Zagrammosoma. Descriptions, distribution maps, host associations, and a key to all known species are provided.

Back pain outcomes after minimally invasive anterior lumbar interbody fusion: a systematic review.

OBJECTIVE: Minimally invasive anterior lumbar interbody fusion surgery (MIS ALIF) is a technique that restores disc height and lumbar lordosis through a smaller exposure and less soft-tissue trauma compared to open approaches. The mini-open and laparoscopic assistance techniques are two main forms of MIS ALIF. The authors conducted a systematic review that sought to critically summarize the literature on back pain following MIS ALIF. METHODS: In March 2020, the authors searched the PubMed, Web of Science, and Cochrane Library databases for studies describing back pain visual analog scale (VAS) outcomes after MIS ALIF. The following exclusion criteria were applied to studies evaluated in full text: 1) the study included fewer than 20 patients, 2) the mean follow-up duration was shorter than 12 months, 3) the study did not report back pain VAS score as an outcome measure, and 4) MIS ALIF was not studied specifically. The methodology for the included studies were evaluated for potential biases and assigned a level of evidence. RESULTS: There were a total of 552 patients included from 13 studies. The most common biases were selection and interviewer bias. The majority of studies were retrospective. The mean sample size was 42.3 patients. The mean follow-up duration was approximately 41.8 months. The mean postoperative VAS reduction was 5.1 points. The mean VAS reduction for standalone grafts was 5.9 points, and 5.0 points for those augmented with posterior fixation. The most common complications included bladder or urinary dysfunction, infection, and hardware-related complications. CONCLUSIONS: This was a systematic review of back pain outcomes following MIS ALIF. Back pain VAS score was reduced postoperatively across all studies. The complication rates were low overall. MIS ALIF is safe and effective at reducing back pain in appropriate patient populations.

Domino Reaction Sequence for the Synthesis of [2.2.2]Diazabicycloalkenes and Base-Promoted Cycloreversion to 2-Pyridone Alkaloids.

A new domino reaction sequence for the construction of 2-pyridone structures is reported. The reaction sequence begins with diacetyldiketopiperazine and proceeds via aldol condensation, alkene isomerization, and intramolecular Diels-Alder cycloaddition. The intermediate [2.2.2]diazabicycloalkene cycloadducts can be isolated or can engage in a base-accelerated extrusion of one lactam bridge to provide the 2-pyridone cycloreversion products. The operation leading to pyridone products can occur in one reaction vessel and proceeds at convenient temperatures.

Comparing human papillomavirus vaccine concerns on Twitter: a cross-sectional study of users in Australia, Canada and the UK.

OBJECTIVE: Opposition to human papillomavirus (HPV) vaccination is common on social media and has the potential to impact vaccine coverage. This study aims to conduct an international comparison of the proportions of tweets about HPV vaccines that express concerns, the types of concerns expressed and the social connections among users posting about HPV vaccines in Australia, Canada and the UK. DESIGN: Using a cross-sectional design, an international comparison of English language tweets about HPV vaccines and social connections among Twitter users posting about HPV vaccines between January 2014 and April 2016 was conducted. The Health Belief Model, one of the most widely used theories in health psychology, was used as the basis for coding the types of HPV vaccine concerns expressed on Twitter. SETTING: The content of tweets and the social connections between users who posted tweets about HPV vaccines from Australia, Canada and the UK. POPULATION: 16 789 Twitter users who posted 43 852 tweets about HPV vaccines. MAIN OUTCOME MEASURES: The proportions of tweets expressing concern, the type of concern expressed and the proportions of local and international social connections between users. RESULTS: Tweets expressing concerns about HPV vaccines made up 14.9% of tweets in Canada, 19.4% in Australia and 22.6% in the UK. The types of concerns expressed were similar across the three countries, with concerns related to 'perceived barriers' being the most common. Users expressing concerns about HPV vaccines in each of the three countries had a relatively high proportion of international followers also expressing concerns. CONCLUSIONS: The proportions and types of HPV vaccine concerns expressed on Twitter were similar across the three countries. Twitter users who mostly expressed concerns about HPV vaccines were better connected to international users who shared their concerns compared with users who did not express concerns about HPV vaccines.

Effects over time of self-reported direct and vicarious racial discrimination on depressive symptoms and loneliness among Australian school students.

BACKGROUND: Racism and racial discrimination are increasingly acknowledged as a critical determinant of health and health inequalities. However, patterns and impacts of racial discrimination among children and adolescents remain under-investigated, including how different experiences of racial discrimination co-occur and influence health and development over time. This study examines associations between self-reported direct and vicarious racial discrimination experiences and loneliness and depressive symptoms over time among Australian school students. METHODS: Across seven schools, 142 students (54.2% female), age at T1 from 8 to 15 years old (M = 11.14, SD = 2.2), and from diverse racial/ethnic and migration backgrounds (37.3% born in English-speaking countries as were one or both parents) self-reported racial discrimination experiences (direct and vicarious) and mental health (depressive symptoms and loneliness) at baseline and 9 months later at follow up. A full cross-lagged panel design was modelled using MPLUS v.7 with all variables included at both time points. RESULTS: A cross-lagged effect of perceived direct racial discrimination on later depressive symptoms and on later loneliness was found. As expected, the effect of direct discrimination on both health outcomes was unidirectional as mental health did not reciprocally influence reported racism. There was no evidence that vicarious racial discrimination influenced either depressive symptoms or loneliness beyond the effect of direct racial discrimination. CONCLUSIONS: Findings suggest direct racial discrimination has a persistent effect on depressive symptoms and loneliness among school students over time. Future work to explore associations between direct and vicarious discrimination is required.

Measuring patient satisfaction: three years' data and experience of using a validated patient questionnaire.

A validated patient satisfaction questionnaire for use in sexual health clinics was run throughout the southwest region (the UK) three times over three years (13 clinics in January 2012 and 12/13 clinics in January 2013 and the same 12 clinics again in January 2014). The questionnaire covered aspects of pre-appointment, performance of health care professionals, facilities, privacy, respectful communication, information on leaving and overall experience. Data from 1750 questionnaires are presented with means and 95% confidence intervals. Patients under the age of 25 were less likely to rate care as excellent and/or very good (P = 0.001) compared with those above the age of 25. There was no difference in satisfaction levels according to gender or sexual orientation. Time in clinic was strongly associated with satisfaction levels - those in clinic for over 90 min expressed less satisfaction (P = 0.0005). We hope that other clinics may find it useful to use the validated questionnaire to look at their own performance and compare it against the southwest data. The main value is to identify areas where changes to service delivery could translate into improved practice and improved satisfaction, particularly where performance is identified as less than the 95% confidence intervals.

Aboriginal health promotion through addressing employment discrimination.

The Localities Embracing and Accepting Diversity (LEAD) program aimed to improve the mental health of Aboriginal Victorians by addressing racial discrimination and facilitating social and economic participation. As part of LEAD, Whittlesea Council adopted the Aboriginal Employment Pathways Strategy (AEPS) to increase Aboriginal employment and retention within the organisation. The Aboriginal Cultural Awareness Training Program was developed to build internal cultural competency and skills in recruiting and retaining Aboriginal staff. Analysis of surveys conducted before (pre; n=124) and after (post; n=107) the training program indicated a significant increase in participant understanding across all program objectives and in support of organisational policies to improve Aboriginal recruitment and retention. Participants ended the training with concrete ideas about intended changes, as well as how these changes could be supported by their supervisors and the wider organisation. Significant resources have since been allocated to implementing the AEPS over 5 years. In line with principles underpinning the National Aboriginal and Torres Strait Islander Health Plan 2013-23, particularly the focus on addressing racism as a determinant of health, this paper explores the AEPS and training program as promising approaches to health promotion through addressing barriers to Aboriginal employment. Possible implications for other large organisations are also considered.

Experiences of racism, racial/ethnic attitudes, motivated fairness and mental health outcomes among primary and secondary school students.

While studies investigating the health effects of racial discrimination for children and youth have examined a range of effect modifiers, to date, relationships between experiences of racial discrimination, student attitudes, and health outcomes remain unexplored. This study uniquely demonstrates the moderating effects of vicarious racism and motivated fairness on the association between direct experiences of racism and mental health outcomes, specifically depressive symptoms and loneliness, among primary and secondary school students. Across seven schools, 263 students (54.4% female), ranging from 8 to 17 years old (M = 11.2, SD = 2.2) reported attitudes about other racial/ethnic groups and experiences of racism. Students from minority ethnic groups (determined by country of birth) reported higher levels of loneliness and more racist experiences relative to the majority group students. Students from the majority racial/ethnic group reported higher levels of loneliness and depressive symptoms if they had more friends from different racial/ethnic groups, whereas the number of friends from different groups had no effect on minority students' loneliness or depressive symptoms. Direct experiences of racism were robustly related to higher loneliness and depressive symptoms in multivariate regression models. However, the association with depressive symptoms was reduced to marginal significance when students reported low motivated fairness. Elaborating on the negative health effects of racism in primary and secondary school students provides an impetus for future research and the development of appropriate interventions.

Endoplasmic reticulum-associated secretory proteins Sec20p, Sec39p, and Dsl1p are involved in peroxisome biogenesis.

Two pathways have been identified for peroxisome formation: (i) growth and division and (ii) de novo synthesis. Recent experiments determined that peroxisomes originate at the endoplasmic reticulum (ER). Although many proteins have been implicated in the peroxisome biogenic program, no proteins in the eukaryotic secretory pathway have been identified as having roles in peroxisome formation. Using the yeast Saccharomyces cerevisiae regulatable Tet promoter Hughes clone collection, we found that repression of the ER-associated secretory proteins Sec20p and Sec39p resulted in mislocalization of the peroxisomal matrix protein chimera Pot1p-green fluorescent protein (GFP) to the cytosol. Likewise, the peroxisomal membrane protein chimera Pex3p-GFP localized to tubular-vesicular structures in cells suppressed for Sec20p, Sec39p, and Dsl1p, which form a complex at the ER. Loss of Sec39p attenuated formation of Pex3p-derived peroxisomal structures following galactose induction of Pex3p-GFP expression from the GAL1 promoter. Expression of Sec20p, Sec39p, and Dsl1p was moderately increased in yeast grown under conditions that proliferate peroxisomes, and Sec20p, Sec39p, and Dsl1p were found to cofractionate with peroxisomes and colocalize with Pex3p-monomeric red fluorescent protein under these conditions. Our results show that SEC20, SEC39, and DSL1 are essential secretory genes involved in the early stages of peroxisome assembly, and this work is the first to identify and characterize an ER-associated secretory machinery involved in peroxisome biogenesis.

Characterization of the sterol-binding domain of oxysterol-binding protein (OSBP)-related protein 4 reveals a novel role in vimentin organization.

Oxysterol-binding protein (OSBP) and OSBP-related protein 4 (ORP4; also designated OSBP2 and HLM) are implicated in sterol-transport and/or sensing via binding to protein partners. The aggregation of vimentin by an N-terminal-truncated variant of ORP4 (ORP4S), but not full-length ORP4L, suggested a functional interaction with this intermediate filament. Herein, we identify ORP4 domains that interact with vimentin, and determine how sterols and OSBP influence this activity. In CHO cells, ORP4L co-localized with filamentous vimentin but extensive remodeling of vimentin filaments required mutation of a leucine repeat motif (amino acids 361-382) adjacent to the oxysterol-binding domain. Similarly, the absence of the leucine repeat in ORP4S 418-878 resulted in co-localization with aggregated vimentin filaments, suggesting that both the sterol-binding domain and leucine repeat are involved. Transient expression of OSBP leucine repeat mutants also promoted vimentin aggregation by a mechanism involving heterodimerization with ORP4L. Glutathione S-transferase (GST)-ORP4 380-878 bound vimentin, cholesterol and 25-hydroxycholesterol in vitro. However, sterol-binding or a mutation that ablated sterol-binding did not influence the interaction of GST-ORP4 with vimentin. Thus the sterol-binding domain of ORP4 binds vimentin, cholesterol and oxysterols, and interacts with the filamentous vimentin network.

Oxysterol-binding protein and vesicle-associated membrane protein-associated protein are required for sterol-dependent activation of the ceramide transport protein.

Sphingomyelin (SM) and cholesterol are coregulated metabolically and associate physically in membrane microdomains involved in cargo sorting and signaling. One mechanism for regulation of this metabolic interface involves oxysterol binding protein (OSBP) via high-affinity binding to oxysterol regulators of cholesterol homeostasis and activation of SM synthesis at the Golgi apparatus. Here, we show that OSBP regulation of SM synthesis involves the endoplasmic reticulum (ER)-to-Golgi ceramide transport protein (CERT). RNA interference (RNAi) experiments in Chinese hamster ovary (CHO)-K1 cells revealed that OSBP and vesicle-associated membrane protein-associated protein (VAP) were required for stimulation of CERT-dependent ceramide transport and SM synthesis by 25-hydroxycholesterol and cholesterol depletion in response to cyclodextrin. Additional RNAi experiments in human embryonic kidney 293 cells supported OSBP involvement in oxysterol-activated SM synthesis and also revealed a role for OSBP in basal SM synthesis. Activation of ER-to-Golgi ceramide transport in CHO-K1 cells required interaction of OSBP with the ER and Golgi apparatus, OSBP-dependent Golgi translocation of CERT, and enhanced CERT-VAP interaction. Regulation of CERT by OSBP, sterols, and VAP reveals a novel mechanism for integrating sterol regulatory signals with ceramide transport and SM synthesis in the Golgi apparatus.

Molecular mechanisms and regulation of ceramide transport.

De novo biosynthesis of sphingolipids begins in the endoplasmic reticulum (ER) and continues in the Golgi apparatus and plasma membrane. A crucial step in sphingolipid biosynthesis is the transport of ceramide by vesicular and non-vesicular mechanisms from its site of synthesis in the ER to the Golgi apparatus. The recent discovery of the ceramide transport protein CERT has revealed a novel pathway for the delivery of ceramide to the Golgi apparatus for sphingomyelin (SM) synthesis. In addition to a ceramide-binding START domain, CERT has FFAT (referring to two phenylalanines [FF] in an acidic tract) and pleckstrin homology (PH) domains that recognize the ER integral membrane protein VAMP-associated protein (VAP) and Golgi-associated PtdIns 4-phosphate, respectively. Mechanisms for vectorial transport involving dual-organellar targeting and sites of deposition of ceramide in the Golgi apparatus are proposed. Similar Golgi-ER targeting motifs are also present in the oxysterol-binding protein (OSBP), which regulates ceramide transport and SM synthesis in an oxysterol-dependent manner. Consequently, this emerges as a potential mechanism for integration of sphingolipid and cholesterol metabolism. The identification of organellar targeting motifs in other related lipid-binding/transport proteins indicate that concepts learned from the study of ceramide transport can be applied to other lipid transport processes.

The role of de novo ceramide synthesis in the mechanism of action of the tricyclic xanthate D609.

The cytotoxic effects of several chemotherapeutic drugs have been linked to elevated de novo ceramide biosynthesis. However, the relationship between the intracellular site(s) of ceramide accumulation and cytotoxicity is poorly understood. Here we examined the relationship between the site of ceramide deposition and inhibition of protein translation and induction of apoptosis by the antitumor/antiviral xanthate, D609. In Chinese hamster ovary (CHO)-K1, HEK-293, and NIH-3T3 cells, D609 caused rapid (1-5 min) and sustained eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation followed by apoptosis after 24 h. Concurrently, D609 stimulated de novo ceramide synthesis and increased ceramide mass 2-fold by 2 h in CHO-K1 cells. In D609-treated CHO-K1 cells, sphingomyelin synthesis was stimulated by brefeldin A, and C5-DMB-ceramide transport to the Golgi apparatus was blocked, indicating ceramide accumulation in the endoplasmic reticulum (ER). However, D609-mediated eIF2alpha phosphorylation, inhibition of protein synthesis, and apoptosis in CHO-K1 cells were not attenuated by fumonisin B1 or l-cycloserine. Interestingly, short-chain ceramide promoted eIF2alpha phosphorylation and inhibited protein synthesis in CHO-K1 cells, indicating that the effectiveness of endogenous ceramide could be limited by access to signaling pathways. Thus, expansion of the ER ceramide pool by D609 was not implicated in early (eIF2alpha phosphorylation) or late (apoptotic) cytotoxic events.