Perspective on Improving Environmental Monitoring of Biothreats.

For more than a decade, the United States has performed environmental monitoring by collecting and analyzing air samples for a handful of biological threat agents (BTAs) in order to detect a possible biological attack. This effort has faced numerous technical challenges including timeliness, sampling efficiency, sensitivity, specificity, and robustness. The cost of city-wide environmental monitoring using conventional technology has also been a challenge. A large group of scientists with expertise in bioterrorism defense met to assess the objectives and current efficacy of environmental monitoring and to identify operational and technological changes that could enhance its efficacy and cost-effectiveness, thus enhancing its value. The highest priority operational change that was identified was to abandon the current concept of city-wide environmental monitoring because the operational costs were too high and its value was compromised by low detection sensitivity and other environmental factors. Instead, it was suggested that the focus should primarily be on indoor monitoring and secondarily on special-event monitoring because objectives are tractable and these operational settings are aligned with likelihood and risk assessments. The highest priority technological change identified was the development of a reagent-less, real-time sensor that can identify a potential airborne release and trigger secondary tests of greater sensitivity and specificity for occasional samples of interest. This technological change could be transformative with the potential to greatly reduce operational costs and thereby create the opportunity to expand the scope and effectiveness of environmental monitoring.

Identification of critical amino acids within the nucleoprotein of Tacaribe virus important for anti-interferon activity.

The arenavirus nucleoprotein (NP) can suppress induction of type I interferon (IFN). This anti-IFN activity is thought to be shared by all arenaviruses with the exception of Tacaribe virus (TCRV). To identify the TCRV NP amino acid residues that prevent its IFN-countering ability, we created a series of NP chimeras between residues of TCRV NP and Pichinde virus (PICV) NP, an arenavirus NP with potent anti-IFN function. Chimera NP analysis revealed that a minimal four amino acid stretch derived from PICV NP could impart efficient anti-IFN activity to TCRV NP. Strikingly, the TCRV NP gene cloned and sequenced from viral stocks obtained through National Institutes of Health Biodefense and Emerging Infections (BEI) resources deviated from the reference sequence at this particular four-amino acid region, GPPT (GenBank KC329849) versus DLQL (GenBank NC004293), respectively at residues 389-392. When efficiently expressed in cells through codon-optimization, TCRV NP containing the GPPT residues rescued the antagonistic IFN function. Consistent with cell expression results, TCRV infection did not stimulate an IFNß response early in infection in multiple cells types (e.g. A549, P388D1), and IRF-3 was not translocated to the nucleus in TCRV-infected A549 cells. Collectively, these data suggest that certain TCRV strain variants contain the important NP amino acids necessary for anti-IFN activity.

Microfluidically-unified cell culture, sample preparation, imaging and flow cytometry for measurement of cell signaling pathways with single cell resolution.

We have developed a microfluidic platform that enables, in one experiment, monitoring of signaling events spanning multiple time-scales and cellular locations through seamless integration of cell culture, stimulation and preparation with downstream analysis. A combination of two single-cell resolution techniques-on-chip multi-color flow cytometry and fluorescence imaging provides multiplexed and orthogonal data on cellular events. Automated, microfluidic operation allows quantitatively- and temporally-precise dosing leading to fine time-resolution and improved reproducibility of measurements. The platform was used to profile the toll-like receptor (TLR4) pathway in macrophages challenged with lipopolysaccharide (LPS)-beginning with TLR4 receptor activation by LPS, through intracellular MAPK signaling, RelA/p65 translocation in real time, to TNF-α cytokine production, all in one small macrophage population (< 5000 cells) while using minute reagent volume (540 nL/condition). The platform is easily adaptable to many cell types including primary cells and provides a generic platform for profiling signaling pathways.

TREM-2 (triggering receptor expressed on myeloid cells 2) is a phagocytic receptor for bacteria.

Phagocytosis, which is essential for the immune response to pathogens, is initiated by specific interactions between pathogens and cell surface receptors expressed by phagocytes. This study identifies triggering receptor expressed on myeloid cells 2 (TREM-2) and its signaling counterpart DAP12 as a molecular complex that promotes phagocytosis of bacteria. Expression of TREM-2-DAP12 enables nonphagocytic Chinese hamster ovary cells to internalize bacteria. This function depends on actin cytoskeleton dynamics and the activity of the small guanosine triphosphatases Rac and Cdc42. Internalization also requires src kinase activity and tyrosine phosphorylation. In bone marrow-derived macrophages, phagocytosis is decreased in the absence of DAP12 and can be restored by expression of TREM-2-DAP12. Depletion of TREM-2 inhibits both binding and uptake of bacteria. Finally, TREM-2-dependent phagocytosis is impaired in Syk-deficient macrophages. This study highlights a novel role for TREM-2-DAP12 in the immune response to bacterial pathogens.

Nuclear translocation kinetics of NF-kappaB in macrophages challenged with pathogens in a microfluidic platform.

We have developed a microfluidic platform for real-time imaging of host-pathogen interactions and cellular signaling events. Host cells are immobilized in a controlled environment for optical interrogation of the kinetics and stochasticity of immune response to pathogenic challenges. Here, we have quantitatively measured activation of the toll-like receptor 4 (TLR4) pathway in RAW264.7 murine macrophage-like cells. This was achieved by measuring the cytoplasm-to-nucleus translocation kinetics of a green fluorescent protein fusion construct to the NF-kappaB transcription factor subunit RelA (GFP-RelA). Translocation kinetics in response to live bacteria and purified lipopolysaccharide (LPS) challenges were measured, and this work presents the first demonstration of live imaging of host cell infection on a microfluidic platform with quantitative analysis of an early (<0.5 h from infection) immune signaling event. Our data show that a 1,000x increase in the LPS dose led to a ~10x increase in a host cell activation metric we developed in order to describe NF-kappaB translocation kinetics. Using this metric, live bacteria challenges were assigned an equivalent LPS dose as a first step towards comparing NF-kappaB translocation kinetics between TLR4-only pathway signaling (activated by LPS) and multiple pathway signaling (activated by whole bacteria). The device also contains a unique architecture for capturing and fluidically isolating single host cells for the purpose of differentiating between primary and secondary immune signaling.

Microfluidic-based cell sorting of Francisella tularensis infected macrophages using optical forces.

We have extended the principle of optical tweezers as a noninvasive technique to actively sort hydrodynamically focused cells based on their fluorescence signal in a microfluidic device. This micro fluorescence-activated cell sorter (microFACS) uses an infrared laser to laterally deflect cells into a collection channel. Green-labeled macrophages were sorted from a 40/60 ratio mixture at a throughput of 22 cells/s over 30 min achieving a 93% sorting purity and a 60% recovery yield. To rule out potential photoinduced cell damage during optical deflection, we investigated the response of mouse macrophage to brief exposures (<4 ms) of focused 1064-nm laser light (9.6 W at the sample). We found no significant difference in viability, cell proliferation, activation state, and functionality between infrared-exposed and unexposed cells. Activation state was measured by the phosphorylation of ERK and nuclear translocation of NF-kappaB, while functionality was assessed in a similar manner, but after a lipopolysaccharide challenge. To demonstrate the selective nature of optical sorting, we isolated a subpopulation of macrophages highly infected with the fluorescently labeled pathogen Francisella tularensis subsp. novicida. A total of 10,738 infected cells were sorted at a throughput of 11 cells/s with 93% purity and 39% recovery.

Different isoforms of the C. elegans FGF receptor are required for attraction and repulsion of the migrating sex myoblasts.

The Caenorhabditis elegans FGF receptor, EGL-15, is alternatively-spliced to yield two major isoforms that differ in their extracellular domains. The EGL-15(5A) isoform is necessary for the gonadal chemoattraction of the migrating sex myoblasts (SMs), while the EGL-15(5B) isoform is required for viability. Here we show that 5A is predominantly expressed in the M lineage, which gives rise to the migrating SMs and their sex muscle descendants, while 5B is predominantly expressed in the hypodermis. Tissue-specific expression, however, explains only part of the functional differences between these two receptor isoforms. 5A can carry out the reciprocal essential function of 5B when expressed in the hypodermis, but 5B is incapable of carrying out SM chemoattraction. Our data, therefore, indicate that the structural differences in these two isoforms contribute to their functional differences. Two lines of evidence indicate that the 5B isoform also plays a role in SM migration, implicating it in the repulsion that is observed when the chemoattraction is compromised. Thus, structural differences in the extracellular domains of these two isoforms can specify either attraction to or repulsion from the gonad.

Talking about a revolution: The impact of site-specific recombinases on genetic analyses in mice.

Site-specific recombinase systems (Cre-loxP, Flp-FRT, and phi C31-att) are transforming both forward and reverse genetics in mice. By enabling high-fidelity DNA modifications to be induced in vitro or in vivo, these systems have incited a wave of new biology, advancing our understanding of gene function, genetic relationships, development, and disease.

Alternative splicing affecting a novel domain in the C. elegans EGL-15 FGF receptor confers functional specificity.

Fibroblast growth factor (FGF) receptors trigger a wide variety of cellular responses as diverse as cell migration, cell proliferation and cell differentiation. However, the molecular basis of the specificity of these responses is not well understood. The C. elegans FGF receptor EGL-15 similarly mediates a number of different responses, including transducing a chemoattractive signal and mediating an essential function. Analysis of the migration-specific alleles of egl-15 has identified a novel EGL-15 isoform that provides a molecular explanation for the different phenotypic effects of lesions at this locus. Alternative splicing yields two EGL-15 proteins containing different forms of a domain located within the extracellular region of the receptors immediately after the first IG domain. Neither of these two domain forms is found in any other FGF receptor. We have tested the roles of these EGL-15 receptor isoforms and their two FGF ligands for their signaling specificity. Our analyses demonstrate different physiological functions for the two receptor variants. EGL-15(5A) is required for the response to the FGF chemoattractant that guides the migrating sex myoblasts to their final positions. By contrast, EGL-15(5B) is both necessary and sufficient to elicit the essential function mediated by this receptor.