Background Macrophages are innate immune cells with diverse functional and molecular phenotypes. This diversity is largely unexplored at the level of single-cell proteomes because of the limitations of quantitative single-cell protein analysis. Results To overcome this limitation, we develop SCoPE2, which substantially increases quantitative accuracy and throughput while lowering cost and hands-on time by introducing automated and miniaturized sample preparation. These advances enable us to analyze the emergence of cellular heterogeneity as homogeneous monocytes differentiate into macrophage-like cells in the absence of polarizing cytokines. SCoPE2 quantifies over 3042 proteins in 1490 single monocytes and macrophages in 10 days of instrument time, and the quantified proteins allow us to discern single cells by cell type. Furthermore, the data uncover a continuous gradient of proteome states for the macrophages, suggesting that macrophage heterogeneity may emerge in the absence of polarizing cytokines. Parallel measurements of transcripts by 10× Genomics suggest that our measurements sample 20-fold more protein copies than RNA copies per gene, and thus, SCoPE2 supports quantification with improved count statistics. This allowed exploring regulatory interactions, such as interactions between the tumor suppressor p53, its transcript, and the transcripts of genes regulated by p53. Conclusions Even in a homogeneous environment, macrophage proteomes are heterogeneous. This heterogeneity correlates to the inflammatory axis of classically and alternatively activated macrophages. Our methodology lays the foundation for automated and quantitative single-cell analysis of proteins by mass spectrometry and demonstrates the potential for inferring transcriptional and post-transcriptional regulation from variability across single cells.

Neisseria meningitidis binds factor H (fH), a key regulator of the alternative complement pathway. A ∼29 kD fH-binding protein expressed in the meningococcal outer membrane was identified by mass spectrometry as GNA1870, a lipoprotein currently under evaluation as a broad-spectrum meningococcal vaccine candidate. GNA1870 was confirmed as the fH ligand on intact bacteria by 1) abrogation of fH binding upon deleting GNA1870, and 2) blocking fH binding by anti-GNA1870 mAbs. fH bound to whole bacteria and purified rGNA1870 representing each of the three variant GNA1870 families. We showed that the amount of fH binding correlated with the level of bacterial GNA1870 expression. High levels of variant 1 GNA1870 expression (either by allelic replacement of gna1870 or by plasmid-driven high-level expression) in strains that otherwise were low-level GNA1870 expressers (and bound low amounts of fH by flow cytometry) restored high levels of fH binding. Diminished fH binding to the GNA1870 deletion mutants was accompanied by enhanced C3 binding and increased killing of the mutants. Conversely, high levels of GNA1870 expression and fH binding enhanced serum resistance. Our findings support the hypothesis that inhibiting the binding of a complement down-regulator protein to the neisserial surface by specific Ab may enhance intrinsic bactericidal activity of the Ab, resulting in two distinct mechanisms of Ab-mediated vaccine efficacy. These data provide further support for inclusion of this molecule in a meningococcal vaccine. To reflect the critical function of this molecule, we suggest calling it fH-binding protein.

SUMMARY Programmed DNA rearrangements in the single-celled eukaryote Oxytricha trifallax completely rewire its germline into a somatic nucleus during development. This elaborate, RNA-mediated pathway eliminates noncoding DNA sequences that interrupt gene loci and reorganizes the remaining fragments by inversions and permutations to produce functional genes. Here, we report the Oxytricha germline genome and compare it to the somatic genome to present a global view of its massive scale of genome rearrangements. The remarkably encrypted genome architecture contains >3,500 scrambled genes, as well as >800 predicted germline-limited genes expressed, and some posttranslationally modified, during genome rearrangements. Gene segments for different somatic loci often interweave with each other. Single gene segments can contribute to multiple, distinct somatic loci. Terminal precursor segments from neighboring somatic loci map extremely close to each other, often overlapping. This genome assembly provides a draft of a scrambled genome and a powerful model for studies of genome rearrangement.

Cellular metabolic fluxes are determined by enzyme activities and metabolite abundances. Biochemical approaches reveal the impact of specific substrates or regulators on enzyme kinetics, but do not capture the extent to which metabolite and enzyme concentrations vary across physiological states, and therefore how cellular reactions are regulated. We measured enzyme and metabolite concentrations and metabolic fluxes across 25 steady-state yeast cultures. We then assessed the extent to which flux can be explained by a Michaelis-Menten relationship between enzyme, substrate, product, and potential regulator concentrations. This revealed three new instances of cross-pathway regulation, which we biochemically verified. These included inhibition of pyruvate kinase by citrate, which accumulated and thereby curtailed glycolytic outflow in nitrogen-limited yeast. Overall, substrate concentrations were the strongest driver of the net rates of cellular metabolic reactions, with metabolite concentrations collectively having more than double the physiological impact of enzymes.

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Subsurface lithoautotrophic microbial ecosystems (SLiMEs) under oligotrophic conditions are typically supported by H 2 . Methanogens and sulfate reducers, and the respective energy processes, are thought to be the dominant players and have been the research foci. Recent investigations showed that, in some deep, fluid-filled fractures in the Witwatersrand Basin, South Africa, methanogens contribute <5% of the total DNA and appear to produce sufficient CH 4 to support the rest of the diverse community. This paradoxical situation reflects our lack of knowledge about the in situ metabolic diversity and the overall ecological trophic structure of SLiMEs. Here, we show the active metabolic processes and interactions in one of these communities by combining metatranscriptomic assemblies, metaproteomic and stable isotopic data, and thermodynamic modeling. Dominating the active community are four autotrophic β-proteobacterial genera that are capable of oxidizing sulfur by denitrification, a process that was previously unnoticed in the deep subsurface. They co-occur with sulfate reducers, anaerobic methane oxidizers, and methanogens, which each comprise <5% of the total community. Syntrophic interactions between these microbial groups remove thermodynamic bottlenecks and enable diverse metabolic reactions to occur under the oligotrophic conditions that dominate in the subsurface. The dominance of sulfur oxidizers is explained by the availability of electron donors and acceptors to these microorganisms and the ability of sulfur-oxidizing denitrifiers to gain energy through concomitant S and H 2 oxidation. We demonstrate that SLiMEs support taxonomically and metabolically diverse microorganisms, which, through developing syntrophic partnerships, overcome thermodynamic barriers imposed by the environmental conditions in the deep subsurface.active subsurface environment | metabolic interactions | sulfur-driven autotrophic denitrifiers | syntrophy | inverted biomass pyramid M icroorganisms living in deep-subsurface ecosystems acquire energy through chemosynthesis and carbon from organic or inorganic sources. Whereas heterotrophs use dissolved organic carbon (DOC) transported from the surface and/or produced in situ, detrital organic deposits buried along with the sediments, and hydrocarbons migrating into petroleum reservoirs, chemolithoautotrophs fix dissolved inorganic carbon (DIC). In oligotrophic systems, subsurface lithoautotrophic microbial ecosystems (SLiMEs) (1) that are fueled by H 2 support the occurrence of autotrophic methanogens, acetogens, and sulfate reducers (2-5). These environments can host highly diverse communities, consisting mostly of prokaryotes, but also multicellular microeukaryotes and viral particles (6-13). Due to the limitation of available nutrients and energy substrates in the oligotrophic subsurface, it is reasonable to hypothesize that subsurface inhabitants with diverse functional traits cooperate syntrophically to maximize energy yield SignificanceMicroorganisms are known to live in the deep ...

Hepatitis B viruses are pararetroviruses that contain a partially dsDNA genome and replicate this DNA through an RNA intermediate (the pregenomic RNA, pgRNA) by reverse transcription. Viral assembly begins with the packaging of the pgRNA into nucleocapsids (NCs), with subsequent reverse transcription within NCs converting the pgRNA into the characteristic dsDNA genome. Only NCs containing this dsDNA (the so-called ''mature'' NCs) are enveloped by the viral envelope proteins and secreted as virions; ''immature'' NCs, i.e., those containing pgRNA or immature reverse transcription intermediates, are excluded from virion formation. This phenomenon is thought to be caused by the emergence of an intrinsic maturation signal only on the mature NCs. To define the maturation signal, we have devised a method to separate mature from immature duck hepatitis B virus NCs and have compared them to NCs derived from secreted virions. Detailed mass spectrometric analyses revealed that the core protein from immature NCs was phosphorylated on at least six sites, whereas the core protein from mature NCs and that from secreted virions was entirely dephosphorylated. These results, together with the known requirement of core phosphorylation for pgRNA packaging and DNA synthesis, suggest that the NC undergoes a dynamic change in phosphorylation state to fulfill its multiple roles at different stages of viral replication. Although phosphorylation of the NCs is required for efficient RNA packaging and DNA synthesis by the immature NCs, dephosphorylation of the mature NCs may trigger envelopment and secretion.hepatitis B virus ͉ viral maturation ͉ posttranslational modification ͉ tandem mass spectrometry ͉ vibrational cooling MALDI-Fourier transform MS H epatitis B viruses (HBVs), or hepadnaviruses, are pararetroviruses that replicate their DNA genome via an RNA intermediate (pregenomic RNA, pgRNA) by reverse transcription (1, 2). Similar to classical retroviruses, hepadnaviruses begin assembly with the formation of intracellular nucleocapsid (NC) particles that specifically package viral pgRNA. However, they undergo reverse transcription before membrane envelopment of NCs and secretion as virions, rather than subsequent to entry into new target cells, like classical retroviruses (2, 3). This replication strategy is manifested in the hepadnaviral maturation phenomenon: secreted hepadnaviral virions contain only the mature, double-stranded relaxed circular (or the mature, doublestranded linear) DNA species, whereas intracellular pools of NCs contain a mix of nucleic acid species, including the pgRNA and DNA species from all of the intermediate stages of reverse transcription (2).The maturation phenomenon implies that mature DNAcontaining NCs may be selectively enveloped and secreted. It was proposed that DNA synthesis and NC envelopment may be coupled through a maturation signal (2). Strong support for this maturation signal model has been obtained (4-6), particularly through the use of a synchronized duck HBV (DHBV) replication system (7,8)...

In order that biological meaning may be derived and testable hypotheses may be built from proteomics experiments, assignments of proteins identified by mass spectrometry or other techniques must be supplemented with additional notation, such as information on known protein functions, protein-protein interactions, or biological pathway associations. Collecting, organizing, and interpreting this data often requires the input of experts in the biological field of study, in addition to the time-consuming search for and compilation of information from online protein databases. Furthermore, visualizing this bulk of information can be challenging due to the limited availability of easy-to-use and freely available tools for this process. In response to these constraints, we have undertaken the design of software to automate annotation and visualization of proteomics data in order to accelerate the pace of research. Here we present the Software Tool for Researching Annotations of Proteins (STRAP) -a user-friendly, open-source C# application. STRAP automatically obtains gene ontology (GO) terms associated with proteins in a proteomics results ID list using the freely accessible UniProtKB and EBI GOA databases. Summarized in an easy-tonavigate tabular format, STRAP includes meta-information on the protein in addition to complimentary GO terminology. Additionally, this information can be edited by the user so that inhouse expertise on particular proteins may be integrated into the larger dataset. STRAP provides a sortable tabular view for all terms, as well as graphical representations of GO-term association data in pie (biological process, cellular component and molecular function) and bar charts (cross comparison of sample sets) to aid in the interpretation of large datasets and differential analyses experiments. Furthermore, proteins of interest may be exported as a unique FASTA-formatted file to allow for customizable re-searching of mass spectrometry data, and gene names corresponding to the proteins in the lists may be encoded in the Gaggle microformat for further characterization, including pathway analysis. STRAP, a tutorial, and the C# source code are freely available from http://cpctools.sourceforge.net.