Functional single-cell analysis of T-cell activation by supported lipid bilayer-tethered ligands on arrays of nanowells

Supported lipid bilayers are an important biomolecular tool for characterizing immunological synapses. Immobilized bilayers presenting tethered ligands on planar substrates have yielded both spatio-temporal and structural insights into how T cell receptors (TCRs) reorganize during the initial formation of synapses upon recognition of peptide antigens bound to major histocompatibility complex (MHC) molecules. The prototypical configuration of these assays, however, limits the extent to which the kinetics and structure of the supramolecular activation clusters of the synapse (that occur in seconds or minutes) can be related to subsequent complex cellular responses, such as cytokine secretion and proliferation, occurring over hours to days. Here we describe a new method that allows correlative measures of both attributes with single-cell resolution by using immobilized lipid bilayers and tethered ligands on the surface of dense arrays of subnanoliter wells. This modification allows each nanowell to function as an artificial antigen-presenting cell (APC), and the synapses formed upon contact can be imaged by fluorescence microscopy. We show that the lipid bilayers remain stable and mobile on the surface of the PDMS, and that modifying the ligands tethered to the bilayer alters the structure of the resulting synapses in expected ways. Finally, we demonstrate that this approach allows the subsequent characterization of secreted cytokines from the activated human T cell clones by microengraving in both antigen- and pan-specific manners. This new technique should allow detailed investigations on how biophysical and structural aspects of the synapse influence the activation of individual T cells and their complex functional responses.National Institute of Allergy and Infectious Diseases (U.S.) (5P01AI045757)National Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051

Early queen infection shapes developmental dynamics and induces long-term disease protection in incipient ant colonies

Infections early in life can have enduring effects on an organism's development and immunity. In this study, we show that this equally applies to developing ‘superorganisms’––incipient social insect colonies. When we exposed newly mated Lasius niger ant queens to a low pathogen dose, their colonies grew more slowly than controls before winter, but reached similar sizes afterwards. Independent of exposure, queen hibernation survival improved when the ratio of pupae to workers was small. Queens that reared fewer pupae before worker emergence exhibited lower pathogen levels, indicating that high brood rearing efforts interfere with the ability of the queen's immune system to suppress pathogen proliferation. Early-life queen pathogen exposure also improved the immunocompetence of her worker offspring, as demonstrated by challenging the workers to the same pathogen a year later. Transgenerational transfer of the queen's pathogen experience to her workforce can hence durably reduce the disease susceptibility of the whole superorganism

Co-founding ant queens prevent disease by performing prophylactic undertaking behaviour

Abstract Background Social insects form densely crowded societies in environments with high pathogen loads, but have evolved collective defences that mitigate the impact of disease. However, colony-founding queens lack this protection and suffer high rates of mortality. The impact of pathogens may be exacerbated in species where queens found colonies together, as healthy individuals may contract pathogens from infectious co-founders. Therefore, we tested whether ant queens avoid founding colonies with pathogen-exposed conspecifics and how they might limit disease transmission from infectious individuals. Results Using Lasius niger queens and a naturally infecting fungal pathogen Metarhizium brunneum, we observed that queens were equally likely to found colonies with another pathogen-exposed or sham-treated queen. However, when one queen died, the surviving individual performed biting, burial and removal of the corpse. These undertaking behaviours were performed prophylactically, i.e. targeted equally towards non-infected and infected corpses, as well as carried out before infected corpses became infectious. Biting and burial reduced the risk of the queens contracting and dying from disease from an infectious corpse of a dead co-foundress. Conclusions We show that co-founding ant queens express undertaking behaviours that, in mature colonies, are performed exclusively by workers. Such infection avoidance behaviours act before the queens can contract the disease and will therefore improve the overall chance of colony founding success in ant queens

2-Propyl 3,3-dibromo-2-hydroxy­pyrrolidine-1-carboxyl­ate

The title compound, C8H13Br2NO3, crystallizes as a non-merohedral twin with twin law −0.6 0 0.4/0 − 1 0 /1.6 0 0.6, and the structure has a refined twin domain ratio of 0.546 (5). The structure shows a compact conformation, with the ester unit roughly coplanar with a mean plane fitted through the non-H atoms of the pyrrolidine ring [dihedral angle = 8.23 (9)°]. In the crystal, inversion dimers linked by pairs of O—H⋯O hydrogen bonds generate an R 2 2(12) motif

Destructive disinfection of infected brood prevents systemic disease spread in ant colonies

Social insects protect their colonies from infectious disease through collective defences that result in social immunity. In ants, workers first try to prevent infection of colony members. Here, we show that if this fails and a pathogen establishes an infection, ants employ an efficient multicomponent behaviour − "destructive disinfection" − to prevent further spread of disease through the colony. Ants specifically target infected pupae during the pathogen's non-contagious incubation period, relying on chemical 'sickness cues' emitted by pupae. They then remove the pupal cocoon, perforate its cuticle and administer antimicrobial poison, which enters the body and prevents pathogen replication from the inside out. Like the immune system of a body that specifically targets and eliminates infected cells, this social immunity measure sacrifices infected brood to stop the pathogen completing its lifecycle, thus protecting the rest of the colony. Hence, the same principles of disease defence apply at different levels of biological organisation

4-(Piperidin-1-yl)-4H-benzo[b]tetra­zolo[1,5-d][1,4]diazepin-5(6H)-one

There are two crystallographically unique mol­ecules present in the asymmetric unit of the title compound, C14H16N6O; in both mol­ecules, the seven-membered diazepinone ring adopts a boat-like conformation and the chair conformation piperidine ring is an axial substituent on the diazepinone ring. In the crystal, each mol­ecule forms hydrogen bonds with its respective symmetry equivalents. Hydrogen bonding between mol­ecule A and symmetry equivalents forms two ring motifs, the first formed by inversion-related N—H⋯O inter­actions and the second formed by C—H⋯O and C—H⋯N inter­actions. The combination of both ring motifs results in the formation of an infinite double tape, which propagates in the a-axis direction. Hydrogen bonding between mol­ecule B and symmetry equivalents forms one ring motif by inversion-related N—H⋯O inter­actions and a second ring motif by C—H⋯O inter­actions, which propagate as a single tape parallel with the c axis

Akt and SHIP Modulate Francisella Escape from the Phagosome and Induction of the Fas-Mediated Death Pathway

Francisella tularensis infects macrophages and escapes phago-lysosomal fusion to replicate within the host cytosol, resulting in host cell apoptosis. Here we show that the Fas-mediated death pathway is activated in infected cells and correlates with escape of the bacterium from the phagosome and the bacterial burden. Our studies also demonstrate that constitutive activation of Akt, or deletion of SHIP, promotes phago-lysosomal fusion and limits bacterial burden in the host cytosol, and the subsequent induction of Fas expression and cell death. Finally, we show that phagosomal escape/intracellular bacterial burden regulate activation of the transcription factors sp1/sp3, leading to Fas expression and cell death. These data identify for the first time host cell signaling pathways that regulate the phagosomal escape of Francisella, leading to the induction of Fas and subsequent host cell death

Evidence for biphasic uncoating during HIV-1 infection from a novel imaging assay

BACKGROUND: Uncoating of the HIV-1 core plays a critical role during early post-fusion stages of infection but is poorly understood. Microscopy-based assays are unable to easily distinguish between intact and partially uncoated viral cores. RESULTS: In this study, we used 5-ethynyl uridine (EU) to label viral-associated RNA during HIV production. At early time points after infection with EU-labeled virions, the viral-associated RNA was stained with an EU-specific dye and was detected by confocal microscopy together with viral proteins. We observed that detection of the viral-associated RNA was specific for EU-labeled virions, was detected only after viral fusion with target cells, and occurred after an initial opening of the core. In vitro staining of cores showed that the opening of the core allowed the small molecule dye, but not RNase A or antibodies, inside. Also, staining of the viral-associated RNA, which is co-localized with nucleocapsid, decays over time after viral infection. The decay rate of RNA staining is dependent on capsid (CA) stability, which was altered by CA mutations or a small molecule inducer of HIV-1 uncoating. While the staining of EU-labeled RNA was not affected by inhibition of reverse transcription, the kinetics of core opening of different CA mutants correlated with initiation of reverse transcription. Analysis of the E45A CA mutant suggests that initial core opening is independent of complete capsid disassembly. CONCLUSIONS: Taken together, our results establish a novel RNA accessibility-based assay that detects an early event in HIV-1 uncoating and can be used to further define this process

Inadvertent VFR-into-IMC Flights: A Qualitative Approach to Describing GA Pilots' First-Hand Experiences

The phenomenon of encountering instrument meteorological conditions (IMC) on a visual flight rules (VFR) flight has been the focus of several previous studies. Most of these past studies, though, have involved examining various databases quantitatively or via a mixed-methods approach in search of possible causal factors such as pilot characteristics, weather conditions, aircraft type, and time of day. Missing from the literature are qualitative studies that tell the story of pilots who actually experienced such flights. To help fill this gap in the aviation literature, the purpose of the current study was to describe the first-hand experiences of GA pilots who inadvertently flew VFR-into-IMC. Participants consisted of 11 male pilots who previously had flown from VFR-into-IMC inadvertently at some point during their aviation career. The study used a phenomenological approach to describe participants’ shared experiences and then applied grounded theory to develop a set of conjectures derived inductively from participants' responses. Using Spradley's (1979) domain analysis to categorize common themes and patterns, the major domains of Weather Considerations and Expectations, Thoughts and Actions, and Postflight Experiences emerged. Major findings from the first domain revealed that as part of their preflight actions prior to departure, participants received a weather briefing, gave little consideration to overall weather conditions, neither expected nor anticipated IMC, and used a variety of communication resources to keep current with weather related issues. Major findings from the second domain revealed that participants recognized changes in the weather en route, used various communication resources to assess their current condition, reacted to IMC by trying to avoid and escape it, expressed feelings of trepidation about what they should do, were surprised over how the weather was not what they expected, and reverted to their training to get out of IMC. Major findings from the third domain revealed that participants' postflight actions ranged from doing nothing to submitting a report to NASA's ASRS, and that lessons learned included acquiring a heightened sense of situational awareness, a need to do a better job in alternative planning, and a greater appreciation for the weather. A comparison of these findings to past studies and theory are discussed, and implications and recommendations for practice and research are provided