In situ architecture of the ER-mitochondria encounter structure.

The endoplasmic reticulum and mitochondria are main hubs of eukaryotic membrane biogenesis that rely on lipid exchange via membrane contact sites1-3, but the underpinning mechanisms remain poorly understood. In yeast, tethering and lipid transfer between the two organelles is mediated by the endoplasmic reticulum-mitochondria encounter structure (ERMES), a four-subunit complex of unresolved stoichiometry and architecture4-6. Here we determined the molecular organization of ERMES within Saccharomyces cerevisiae cells using integrative structural biology by combining quantitative live imaging, cryo-correlative microscopy, subtomogram averaging and molecular modelling. We found that ERMES assembles into approximately 25 discrete bridge-like complexes distributed irregularly across a contact site. Each bridge consists of three synaptotagmin-like mitochondrial lipid binding protein domains oriented in a zig-zag arrangement. Our molecular model of ERMES reveals a pathway for lipids. These findings resolve the in situ supramolecular architecture of a major inter-organelle lipid transfer machinery and provide a basis for the mechanistic understanding of lipid fluxes in eukaryotic cells.

Structural basis of TRAPPIII-mediated Rab1 activation.

The GTPase Rab1 is a master regulator of the early secretory pathway and is critical for autophagy. Rab1 activation is controlled by its guanine nucleotide exchange factor, the multisubunit TRAPPIII complex. Here, we report the 3.7 Å cryo-EM structure of the Saccharomyces cerevisiae TRAPPIII complex bound to its substrate Rab1/Ypt1. The structure reveals the binding site for the Rab1/Ypt1 hypervariable domain, leading to a model for how the complex interacts with membranes during the activation reaction. We determined that stable membrane binding by the TRAPPIII complex is required for robust activation of Rab1/Ypt1 in vitro and in vivo, and is mediated by a conserved amphipathic α-helix within the regulatory Trs85 subunit. Our results show that the Trs85 subunit serves as a membrane anchor, via its amphipathic helix, for the entire TRAPPIII complex. These findings provide a structural understanding of Rab activation on organelle and vesicle membranes.

A SURF4-to-proteoglycan relay mechanism that mediates the sorting and secretion of a tagged variant of sonic hedgehog.

SignificanceSonic Hedgehog (Shh) is a key signaling molecule that plays important roles in embryonic patterning, cell differentiation, and organ development. Although fundamentally important, the molecular mechanisms that regulate secretion of newly synthesized Shh are still unclear. Our study reveals a role for the cargo receptor, SURF4, in facilitating export of Shh from the endoplasmic reticulum (ER) via a ER export signal. In addition, our study provides evidence suggesting that proteoglycans promote the dissociation of SURF4 from Shh at the Golgi, suggesting a SURF4-to-proteoglycan relay mechanism. These analyses provide insight into an important question in cell biology: how do cargo receptors capture their clients in one compartment, then disengage at their destination?

Selective inhibition of protein secretion by abrogating receptor-coat interactions during ER export.

Protein secretion is an essential process that drives cell growth, movement, and communication. Protein traffic within the secretory pathway occurs via transport intermediates that bud from one compartment and fuse with a downstream compartment to deliver their contents. Here, we explore the possibility that protein secretion can be selectively inhibited by perturbing protein-protein interactions that drive capture into transport vesicles. Human proprotein convertase subtilisin/kexin type 9 (PCSK9) is a determinant of cholesterol metabolism whose secretion is mediated by a specific cargo adaptor protein, SEC24A. We map a series of protein-protein interactions between PCSK9, its endoplasmic reticulum (ER) export receptor SURF4, and SEC24A that mediate secretion of PCSK9. We show that the interaction between SURF4 and SEC24A can be inhibited by 4-phenylbutyrate (4-PBA), a small molecule that occludes a cargo-binding domain of SEC24. This inhibition reduces secretion of PCSK9 and additional SURF4 clients that we identify by mass spectrometry, leaving other secreted cargoes unaffected. We propose that selective small-molecule inhibition of cargo recognition by SEC24 is a potential therapeutic intervention for atherosclerosis and other diseases that are modulated by secreted proteins.

Evolutionary balance between foldability and functionality of a glucose transporter.

Despite advances in resolving the structures of multi-pass membrane proteins, little is known about the native folding pathways of these complex structures. Using single-molecule magnetic tweezers, we here report a folding pathway of purified human glucose transporter 3 (GLUT3) reconstituted within synthetic lipid bilayers. The N-terminal major facilitator superfamily (MFS) fold strictly forms first, serving as a structural template for its C-terminal counterpart. We found polar residues comprising the conduit for glucose molecules present major folding challenges. The endoplasmic reticulum membrane protein complex facilitates insertion of these hydrophilic transmembrane helices, thrusting GLUT3's microstate sampling toward folded structures. Final assembly between the N- and C-terminal MFS folds depends on specific lipids that ease desolvation of the lipid shells surrounding the domain interfaces. Sequence analysis suggests that this asymmetric folding propensity across the N- and C-terminal MFS folds prevails for metazoan sugar porters, revealing evolutionary conflicts between foldability and functionality faced by many multi-pass membrane proteins.

An in vitro vesicle formation assay reveals cargo clients and factors that mediate vesicular trafficking.

The fidelity of protein transport in the secretory pathway relies on the accurate sorting of proteins to their correct destinations. To deepen our understanding of the underlying molecular mechanisms, it is important to develop a robust approach to systematically reveal cargo proteins that depend on specific sorting machinery to be enriched into transport vesicles. Here, we used an in vitro assay that reconstitutes packaging of human cargo proteins into vesicles to quantify cargo capture. Quantitative mass spectrometry (MS) analyses of the isolated vesicles revealed cytosolic proteins that are associated with vesicle membranes in a GTP-dependent manner. We found that two of them, FAM84B (also known as LRAT domain containing 2 or LRATD2) and PRRC1, contain proline-rich domains and regulate anterograde trafficking. Further analyses revealed that PRRC1 is recruited to endoplasmic reticulum (ER) exit sites, interacts with the inner COPII coat, and its absence increases membrane association of COPII. In addition, we uncovered cargo proteins that depend on GTP hydrolysis to be captured into vesicles. Comparing control cells with cells depleted of the cargo receptors, SURF4 or ERGIC53, we revealed specific clients of each of these two export adaptors. Our results indicate that the vesicle formation assay in combination with quantitative MS analysis is a robust and powerful tool to uncover novel factors that mediate vesicular trafficking and to uncover cargo clients of specific cellular factors.

The Attachment Versus Differentiation Debate: Bringing the Conversation to Parent-Child Relationships.

This article contributes to the attachment versus differentiation debate, bringing the conversation to parent-child relationships. While attachment theory's (AT) approach emphasizes bonding, Bowen family systems theory's (BFST) differentiation approach emphasizes emotional boundaries. They both suggest that balancing autonomy and connection is important, but AT conceptualizes this in terms of the parent's ability to meet the child's needs for autonomy and connection, while BFST conceptualizes this in terms of the parent's and child's ability to be connected due to mutual respect for each other's emotional boundaries. They similarly recognize that: (1) emotionally mature parents respect children individuality, (2) emotionally immature parents may project their needs and wishes onto children, and (3) emotionally mature parents focus on calming themselves to help their children to be calm. They differ in that: (1) BFST suggests that children may project their needs and wishes onto their parents and intrude on their parents' emotional boundaries, and AT does not conceptualize this; (2) BFST suggests that caregiver over-involvement may be experienced as positive for a child and program them to be excessively needy, and AT suggests that caregiver over-involvement is negative for children and neediness is caused by under-involved caregiving; and (3) BFST suggests that therapists should not try to be a parent to their clients as this can replicate the fusion that the client experienced with their parents, and AT suggests that therapists should try to be like a good parent to their clients to help them to develop more secure attachment styles.

The Ubp3/Bre5 deubiquitylation complex modulates COPII vesicle formation.

The appropriate delivery of secretory proteins to the correct subcellular destination is an essential cellular process. In the endoplasmic reticulum (ER), secretory proteins are captured into COPII vesicles that generally exclude ER resident proteins and misfolded proteins. We previously characterized a collection of yeast mutants that fail to enforce this sorting stringency and improperly secrete the ER chaperone, Kar2 (Copic et al., Genetics 2009). Here, we used the emp24Δ mutant strain that secretes Kar2 to identify candidate proteins that might regulate ER export, reasoning that loss of regulatory proteins would restore sorting stringency. We find that loss of the deubiquitylation complex Ubp3/Bre5 reverses all of the known phenotypes of the emp24Δ mutant, and similarly reverses Kar2 secretion of many other ER retention mutants. Based on a combination of genetic interactions and live cell imaging, we conclude that Ubp3 and Bre5 modulate COPII coat assembly at ER exit sites. Therefore, we propose that Ubp3/Bre5 influences the rate of vesicle formation from the ER that in turn can impact ER quality control events.

Ribosome-associated quality control of membrane proteins at the endoplasmic reticulum.

Protein synthesis is an energetically costly, complex and risky process. Aberrant protein biogenesis can result in cellular toxicity and disease, with membrane-embedded proteins being particularly challenging for the cell. In order to protect the cell from consequences of defects in membrane proteins, quality control systems act to maintain protein homeostasis. The majority of these pathways act post-translationally; however, recent evidence reveals that membrane proteins are also subject to co-translational quality control during their synthesis in the endoplasmic reticulum (ER). This newly identified quality control pathway employs components of the cytosolic ribosome-associated quality control (RQC) machinery but differs from canonical RQC in that it responds to biogenesis state of the substrate rather than mRNA aberrations. This ER-associated RQC (ER-RQC) is sensitive to membrane protein misfolding and malfunctions in the ER insertion machinery. In this Review, we discuss the advantages of co-translational quality control of membrane proteins, as well as potential mechanisms of substrate recognition and degradation. Finally, we discuss some outstanding questions concerning future studies of ER-RQC of membrane proteins.

Comparison of Antimicrobial-Resistant Escherichia coli Isolates from Urban Raccoons and Domestic Dogs.

Wildlife can be exposed to antimicrobial-resistant bacteria (ARB) via multiple pathways. Spatial overlap with domestic animals is a prominent exposure pathway. However, most studies of wildlife-domestic animal interfaces have focused on livestock and little is known about the wildlife-companion animal interface. Here, we investigated the prevalence and phylogenetic relatedness of extended-spectrum cephalosporin-resistant (ESC-R) Escherichia coli from raccoons (Procyon lotor) and domestic dogs (Canis lupus familiaris) in the metropolitan area of Chicago, IL, USA. To assess the potential importance of spatial overlap with dogs, we explored whether raccoons sampled at public parks (i.e., parks where people and dogs could enter) differed in prevalence and phylogenetic relatedness of ESC-R E. coli to raccoons sampled at private parks (i.e., parks where people and dogs could not enter). Raccoons had a significantly higher prevalence of ESC-R E. coli (56.9%) than dogs (16.5%). However, the richness of ESC-R E. coli did not vary by host species. Further, core single-nucleotide polymorphism (SNP)-based phylogenetic analyses revealed that isolates did not cluster by host species, and in some cases displayed a high degree of similarity (i.e., differed by less than 20 core SNPs). Spatial overlap analyses revealed that ESC-R E. coli were more likely to be isolated from raccoons at public parks than raccoons at private parks, but only for parks located in suburban areas of Chicago, not urban areas. That said, ESC-R E. coli isolated from raccoons did not genetically cluster by park of origin. Our findings suggest that domestic dogs and urban/suburban raccoons can have a diverse range of ARB, some of which display a high degree of genetic relatedness (i.e., differ by less than 20 core SNPs). Given the differences in prevalence, domestic dogs are unlikely to be an important source of exposure for mesocarnivores in urbanized areas. IMPORTANCE Antimicrobial-resistant bacteria (ARB) have been detected in numerous wildlife species across the globe, which may have important implications for human and animal health. Wildlife can be exposed to ARB via numerous pathways, including via spatial overlap with domestic animals. However, the interface with domestic animals has mostly been explored for livestock and little is known about the interface between wild animals and companion animals. Our work suggests that urban and suburban wildlife can have similar ARB to local domestic dogs, but local dogs are unlikely to be a direct source of exposure for urban-adapted wildlife. This finding is important because it underscores the need to incorporate wildlife into antimicrobial resistance surveillance efforts, and to investigate whether certain urban wildlife species could act as additional epidemiological pathways of exposure for companion animals, and indirectly for humans.

Merging Metagenomics and Spatial Epidemiology To Understand the Distribution of Antimicrobial Resistance Genes from Enterobacteriaceae in Wild Owls.

Antimicrobial resistance (AMR) is a well-documented phenomenon in bacteria from many natural ecosystems, including wild animals. However, the specific determinants and spatial distribution of resistant bacteria and antimicrobial resistance genes (ARGs) in the environment remain incompletely understood. In particular, information regarding the importance of anthropogenic sources of AMR relative to that of other biological and ecological influences is lacking. We conducted a cross-sectional study of AMR in great horned owls (Bubo virginianus) and barred owls (Strix varia) admitted to a rehabilitation center in the midwestern United States. A combination of selective culture enrichment and shotgun metagenomic sequencing was used to identify ARGs from Enterobacteriaceae Overall, the prevalence of AMR was comparable to that in past studies of resistant Enterobacteriaceae in raptors, with acquired ARGs being identified in 23% of samples. Multimodel regression analyses identified seasonality and owl age to be important predictors of the likelihood of the presence of ARGs, with birds sampled during warmer months being more likely to harbor ARGs than those sampled during cooler months and with birds in their hatch year being more likely to harbor ß-lactam ARGs than adults. Beyond host-specific determinants, ARG-positive owls were also more likely to be recovered from areas of high agricultural land cover. Spatial clustering analyses identified a significant high-risk cluster of tetracycline resistance gene-positive owls in the southern sampling range, but this could not be explained by any predictor variables. Taken together, these results highlight the complex distribution of AMR in natural environments and suggest that both biological and anthropogenic factors play important roles in determining the emergence and persistence of AMR in wildlife.IMPORTANCE Antimicrobial resistance (AMR) is a multifaceted problem that poses a worldwide threat to human and animal health. Recent reports suggest that wildlife may play an important role in the emergence, dissemination, and persistence of AMR. As such, there have been calls for better integration of wildlife into current research on AMR, including the use of wild animals as biosentinels of AMR contamination in the environment. A One Health approach can be used to gain a better understanding of all AMR sources and pathways, particularly those at the human-animal-environment interface. Our study focuses on this interface in order to assess the effect of human-impacted landscapes on AMR in a wild animal. This work highlights the value of wildlife rehabilitation centers for environmental AMR surveillance and demonstrates how metagenomic sequencing within a spatial epidemiology framework can be used to address questions surrounding AMR complexity in natural ecosystems.

Genomic Landscape of Ornithobacterium rhinotracheale in Commercial Turkey Production in the United States.

Ornithobacterium rhinotracheale is a causative agent of respiratory tract infections in avian hosts worldwide but is a particular problem for commercial turkey production. Little is known about the ecologic and evolutionary dynamics of O. rhinotracheale, which makes prevention and control of this pathogen a challenge. The purpose of this study was to gain insight into the genetic relationships between O. rhinotracheale populations through comparative genomics of clinical isolates from different U.S. turkey producers. O. rhinotracheale clinical isolates were collected from four major U.S. turkey producers and several independent turkey growers from the upper Midwest and Southeast, and whole-genome sequencing was performed. Genomes were compared phylogenetically using single nucleotide polymorphism (SNP)-based analysis, and then assembly and annotations were performed to identify genes encoding putative virulence factors and antimicrobial resistance determinants. A pangenome approach was also used to establish a core set of genes consistently present in O. rhinotracheale and to highlight differences in gene content between phylogenetic clades. A total of 1,457 nonrecombinant SNPs were identified from 157 O. rhinotracheale genomes, and four distinct phylogenetic clades were identified. Isolates clustered by company on the phylogenetic tree, however, and each company had isolates in multiple clades with similar collection dates, indicating that there are multiple O. rhinotracheale strains circulating within each of the companies examined. Additionally, several antimicrobial resistance proteins, putative virulence factors, and the pOR1 plasmid were associated with particular clades and multilocus sequence types, which may explain why the same strains seem to have persisted in the same turkey operations for decades.IMPORTANCE The whole-genome approach enhances our understanding of evolutionary relationships between clinical Ornithobacterium rhinotracheale isolates from different commercial turkey producers and allows for identification of genes associated with virulence, antimicrobial resistance, or mobile genetic elements that are often excluded using traditional typing methods. Additionally, differentiating O. rhinotracheale isolates at the whole-genome level may provide insight into selection of the most appropriate autogenous vaccine strain, or groups of strains, for a given population of clinical isolates.

Microbial associations and spatial proximity predict North American moose (Alces alces) gastrointestinal community composition.

Microbial communities are increasingly recognized as crucial for animal health. However, our understanding of how microbial communities are structured across wildlife populations is poor. Mechanisms such as interspecific associations are important in structuring free-living communities, but we still lack an understanding of how important interspecific associations are in structuring gut microbial communities in comparison with other factors such as host characteristics or spatial proximity of hosts. Here, we ask how gut microbial communities are structured in a population of North American moose Alces alces. We identify key microbial interspecific associations within the moose gut and quantify how important they are relative to key host characteristics, such as body condition, for predicting microbial community composition. We sampled gut microbial communities from 55 moose in a population experiencing decline due to a myriad of factors, including pathogens and malnutrition. We examined microbial community dynamics in this population utilizing novel graphical network models that can explicitly incorporate spatial information. We found that interspecific associations were the most important mechanism structuring gut microbial communities in moose and detected both positive and negative associations. Models only accounting for associations between microbes had higher predictive value compared to models including moose sex, evidence of previous pathogen exposure or body condition. Adding spatial information on moose location further strengthened our model and allowed us to predict microbe occurrences with ~90% accuracy. Collectively, our results suggest that microbial interspecific associations coupled with host spatial proximity are vital in shaping gut microbial communities in a large herbivore. In this case, previous pathogen exposure and moose body condition were not as important in predicting gut microbial community composition. The approach applied here can be used to quantify interspecific associations and gain a more nuanced understanding of the spatial and host factors shaping microbial communities in non-model hosts.

Training Psychologists in Integrated Primary Care and Child Maltreatment: Trainee and Supervisor Perspectives on Lessons Learned.

Psychologists are key team members in the delivery of integrated behavioral healthcare. Healthcare reform has supported a shift toward a team-based, interdisciplinary model of service delivery, with increasing emphasis on primary care services, prevention, and health promotion. In conjunction with this shift has been a greater focus on psychosocial problems and social determinants of health, particularly childhood adversity. Psychologists in primary care are uniquely positioned to advance efforts to prevent and ameliorate childhood adversity, which are essential to improving care for underserved populations and reducing health disparities. Targeted training efforts are needed to increase the number of psychologists equipped to work in primary care settings with underserved populations. This paper provides an overview of a training program designed to provide psychology trainees with specialized training in both integrated primary care and child maltreatment. The overarching goal of the program is to provide trainees with the skillset to work within integrated primary care settings and the expertise needed to further efforts to address and prevent child maltreatment, as well as childhood adversity more broadly, to improve outcomes for underserved populations. The paper reviews strengths, challenges, and lessons learned from this program.

Two novel effectors of trafficking and maturation of the yeast plasma membrane H+ -ATPase.

The endoplasmic reticulum (ER) is the entry site of proteins into the endomembrane system. Proteins exit the ER via coat protein II (COPII) vesicles in a selective manner, mediated either by direct interaction with the COPII coat or aided by cargo receptors. Despite the fundamental role of such receptors in protein sorting, only a few have been identified. To further define the machinery that packages secretory cargo and targets proteins from the ER to Golgi membranes, we used multiple systematic approaches, which revealed 2 uncharacterized proteins that mediate the trafficking and maturation of Pma1, the essential yeast plasma membrane proton ATPase. Ydl121c (Exp1) is an ER protein that binds Pma1, is packaged into COPII vesicles, and whose deletion causes ER retention of Pma1. Ykl077w (Psg1) physically interacts with Exp1 and can be found in the Golgi and coat protein I (COPI) vesicles but does not directly bind Pma1. Loss of Psg1 causes enhanced degradation of Pma1 in the vacuole. Our findings suggest that Exp1 is a Pma1 cargo receptor and that Psg1 aids Pma1 maturation in the Golgi or affects its retrieval. More generally our work shows the utility of high content screens in the identification of novel trafficking components.

Phylogenomic Analysis of Extraintestinal Pathogenic Escherichia coli Sequence Type 1193, an Emerging Multidrug-Resistant Clonal Group.

The fluoroquinolone-resistant sequence type 1193 (ST1193) of Escherichia coli, from the ST14 clonal complex (STc14) within phylogenetic group B2, has appeared recently as an important cause of extraintestinal disease in humans. Although this emerging lineage has been characterized to some extent using conventional methods, it has not been studied extensively at the genomic level. Here, we used whole-genome sequence analysis to compare 355 ST1193 isolates with 72 isolates from other STs within STc14. Using core genome phylogeny, the ST1193 isolates formed a tightly clustered clade with many genotypic similarities, unlike ST14 isolates. All ST1193 isolates possessed the same set of three chromosomal mutations conferring fluoroquinolone resistance, carried the fimH64 allele, and were lactose non-fermenting. Analysis revealed an evolutionary progression from K1 to K5 capsular types and acquisition of an F-type virulence plasmid, followed by changes in plasmid structure congruent with genome phylogeny. In contrast, the numerous identified antimicrobial resistance genes were distributed incongruently with the underlying phylogeny, suggesting frequent gain or loss of the corresponding resistance gene cassettes despite retention of the presumed carrier plasmids. Pangenome analysis revealed gains and losses of genetic loci occurring during the transition from ST14 to ST1193 and from the K1 to K5 capsular types. Using time-scaled phylogenetic analysis, we estimated that current ST1193 clades first emerged approximately 25 years ago. Overall, ST1193 appears to be a recently emerged clone in which both stepwise and mosaic evolution have contributed to epidemiologic success.

Assessing Transmission of Antimicrobial-Resistant Escherichia coli in Wild Giraffe Contact Networks.

There is growing evidence that anthropogenic sources of antibiotics and antimicrobial-resistant bacteria can spill over into natural ecosystems, raising questions about the role wild animals play in the emergence, maintenance, and dispersal of antibiotic resistance genes. In particular, we lack an understanding of how resistance genes circulate within wild animal populations, including whether specific host characteristics, such as social associations, promote interhost transmission of these genes. In this study, we used social network analysis to explore the forces shaping population-level patterns of resistant Escherichia coli in wild giraffe (Giraffa camelopardalis) and assess the relative importance of social contact for the dissemination of resistant E. coli between giraffe. Of 195 giraffe sampled, only 5.1% harbored E. coli isolates resistant to one or more tested antibiotics. Whole-genome sequencing on a subset of resistant isolates revealed a number of acquired resistance genes with linkages to mobile genetic elements. However, we found no evidence that the spread of resistance genes among giraffe was facilitated by interhost associations. Giraffe with lower social degree were more likely to harbor resistant E. coli, but this relationship was likely driven by a correlation between an individual's social connectedness and age. Indeed, resistant E. coli was most frequently detected in socially isolated neonates, indicating that resistant E. coli may have a selective advantage in the gastrointestinal tracts of neonates compared to other age classes. Taken together, these results suggest that the maintenance of antimicrobial-resistant bacteria in wild populations may, in part, be determined by host traits and microbial competition dynamics within the host.IMPORTANCE Antimicrobial resistance represents a significant threat to human health, food security, and the global economy. To fully understand the evolution and dissemination of resistance genes, a complete picture of antimicrobial resistance in all biological compartments, including natural ecosystems, is required. The environment and wild animals may act as reservoirs for anthropogenically derived resistance genes that could be transferrable to clinically relevant bacteria of humans and domestic animals. Our study investigated the possible transmission mechanisms for antimicrobial-resistant bacteria within a wild animal population and, more broadly, contributes to our understanding of how resistance genes are spread and maintained in natural ecosystems.

Heparin Decreases in Tumor Necrosis Factor α (TNFα)-induced Endothelial Stress Responses Require Transmembrane Protein 184A and Induction of Dual Specificity Phosphatase 1.

Despite the large number of heparin and heparan sulfate binding proteins, the molecular mechanism(s) by which heparin alters vascular cell physiology is not well understood. Studies with vascular smooth muscle cells (VSMCs) indicate a role for induction of dual specificity phosphatase 1 (DUSP1) that decreases ERK activity and results in decreased cell proliferation, which depends on specific heparin binding. The hypothesis that unfractionated heparin functions to decrease inflammatory signal transduction in endothelial cells (ECs) through heparin-induced expression of DUSP1 was tested. In addition, the expectation that the heparin response includes a decrease in cytokine-induced cytoskeletal changes was examined. Heparin pretreatment of ECs resulted in decreased TNFα-induced JNK and p38 activity and downstream target phosphorylation, as identified through Western blotting and immunofluorescence microscopy. Through knockdown strategies, the importance of heparin-induced DUSP1 expression in these effects was confirmed. Quantitative fluorescence microscopy indicated that heparin treatment of ECs reduced TNFα-induced increases in stress fibers. Monoclonal antibodies that mimic heparin-induced changes in VSMCs were employed to support the hypothesis that heparin was functioning through interactions with a receptor. Knockdown of transmembrane protein 184A (TMEM184A) confirmed its involvement in heparin-induced signaling as seen in VSMCs. Therefore, TMEM184A functions as a heparin receptor and mediates anti-inflammatory responses of ECs involving decreased JNK and p38 activity.

Illuminating the black box of TNF action in tuberculous granulomas.

TNF clearly contributes to immunity to intracellular pathogens, but how it does so is incompletely understood. In this issue of Immunity, Clay et al. (2008) provide unique insights, using intravital microscopy and the zebrafish-embryo model of tuberculosis.

Social Cognition, Child Neglect, and Child Injury Risk: The Contribution of Maternal Social Information Processing to Maladaptive Injury Prevention Beliefs Within a High-Risk Sample.

Objective: Inadequate supervision has been linked to children's injuries. Parental injury prevention beliefs may play a role in supervision, yet little theory has examined the origins of such beliefs. This study examined whether mothers who perpetrated child neglect, who as a group provide inadequate supervision, have more maladaptive beliefs. Then, it tested a social information processing (SIP) model for explaining these beliefs. Methods: SIP and injury prevention beliefs were assessed in disadvantaged mothers of preschoolers (N = 145), half with child neglect histories. Results: The neglect group exhibited significantly more maladaptive injury prevention beliefs than comparisons. As predicted, SIP was linked to beliefs that may increase injury risk, even after accounting for relevant sociodemographic variables. Conclusions: Findings support the link of beliefs to injury risk and suggest that specific cognitive problems may underlie these beliefs. Future work should further validate this model, which may inform enhancements to prevention efforts.