Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.

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• TLR-stimulated macrophages synthesize, release, and hydrolyze ATP via CD39 to regulate their own activation state.• The loss of macrophage CD39 prevents regulatory macrophage development and leads to lethal inflammatory responses and septic shock in mice.Sepsis is a highly fatal disease caused by an initial hyperinflammatory response followed by a state of profound immunosuppression. Although it is well appreciated that the initial production of proinflammatory cytokines by macrophages accompanies the onset of sepsis, it remains unclear what causes the transition to an immunosuppressive state. In this study, we reveal that macrophages themselves are key regulators of this transition and that the surface enzyme CD39 plays a critical role in self-limiting the activation process. We demonstrate that Toll-like receptor (TLR)-stimulated macrophages modulate their activation state by increasing the synthesis and secretion of adenosine triphosphate (ATP). This endogenous ATP is paradoxically immunosuppressive due to its rapid catabolism into adenosine by CD39. Macrophages lacking CD39 are unable to transition to a regulatory state and consequently continue to produce inflammatory cytokines. The importance of this transition is demonstrated in a mouse model of sepsis, where small numbers of CD39-deficient macrophages were sufficient to induce lethal endotoxic shock. Thus, these data implicate CD39 as a key "molecular switch" that allows macrophages to self-limit their activation state. We propose that therapeutics targeting the release and hydrolysis of ATP by macrophages may represent new ways to treat inflammatory diseases. (Blood. 2013;122(11):1935-1945 IntroductionFailure to control inflammatory macrophage activation responses can lead to pathological diseases, best exemplified by sepsis. Despite our growing understanding of its pathogenesis, sepsis continues to affect more than 200 000 people annually in the United States, with a mortality rate as high as 50%.1,2 The severe pathology associated with sepsis occurs in response to the hyperproduction of macrophagederived inflammatory cytokines, which can lead to vascular and tissue destruction, multiple organ failure, shock, and death.3 Intriguingly, macrophages isolated from late-stage septic individuals exhibit the phenotype of immunosuppressive, regulatory macrophages, expressing high levels of the anti-inflammatory cytokine interleukin-10 (IL-10) and low levels of tumor necrosis factor a (TNF-a) and IL-12. [4][5][6] These observations suggest the transition from inflammatory to immunosuppressive macrophages may be critical to control initial inflammatory responses and prevent lethal septic shock. 4,7,8 However, the molecular mechanism by which this transition is achieved remains poorly understood.In the present work, we examine the role that endogenous CD39 and adenosine triphosphate (ATP) play in regulating the macrophage inflammatory response. Recently, extracellular ATP (eATP) has been characterized as a "danger signal" that can promote inflammation through ...

Rev is an essential HIV-1 regulatory protein that binds the Rev responsive element (RRE) within the env gene of the HIV-1 RNA genome and is involved in transport of unspliced or partially spliced viral mRNA from the cell nucleus to the cytoplasm. Previous studies have shown that a short alpha-helical peptide derived from Rev (Rev 34-50), and a truncated form of the RRE sequence provide a useful in vitro system to study this interaction while still preserving the essential aspects of the native complex. We have selectively incorporated the fluorescent probe 2-aminopurine 2'-O-methylriboside (2-AP) into the RRE sequence in nonperturbing positions (A68 and U72) such that the binding of both Rev peptide and aminoglycoside ligands could be characterized directly by fluorescence methods. Rev peptide binding to the RRE-72AP variant resulted in a 2-fold fluorescence increase that provided a useful signal to monitor this binding interaction (K(D) = 20 +/- 7 nM). Using stopped-flow kinetic measurements, we have shown that specific Rev peptide binding occurs by a two-step process involving diffusion-controlled encounter, followed by isomerization of the RNA. Using the RRE-68AP and -72AP constructs, three classes of binding sites for the aminoglycoside neomycin were unambiguously detected. The first site is noninhibitory to Rev binding (K(D) = 0.24 +/- 0.040 microM), the second site inhibited Rev binding in a competitive fashion (K(D) = 1. 8 +/- 0.8 microM), and the third much weaker site (or sites) is attributed to nonspecific binding (K(D) >/= 40 microM). Complementary NMR measurements have shown that neomycin forms both a specific binary complex with RRE and a specific ternary complex with RRE and Rev. NMR data further suggest that neomycin occupies a similar high-affinity binding site in both the binary and ternary complexes, and that this site is located in the lower stem region of RRE.