Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retro-transposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing ∼57% of the genome, reveals 20,486 protein-coding genes and expansions of gene families associated with tick–host interactions. We report insights from genome analyses into parasitic processes unique to ticks, including host ‘questing', prolonged feeding, cuticle synthesis, blood meal concentration, novel methods of haemoglobin digestion, haem detoxification, vitellogenesis and prolonged off-host survival. We identify proteins associated with the agent of human granulocytic anaplasmosis, an emerging disease, and the encephalitis-causing Langat virus, and a population structure correlated to life-history traits and transmission of the Lyme disease agent.

Highlights d Cardiomyocytes release subcellular particles called exophers d Cardiac exophers transport defective mitochondria for elimination d cMacs capture and eliminate exophers though Mertk

The antimicrobial functions of neutrophils are facilitated by a defensive armamentarium of proteins stored in granules, and by the formation of neutrophil extracellular traps (NETs). However, the toxic nature of these structures poses a threat to highly vascularized tissues, such as the lungs. Here, we identified a cell-intrinsic program that modified the neutrophil proteome in the circulation and caused the progressive loss of granule content and reduction of the NET-forming capacity. This program was driven by the receptor CXCR2 and by regulators of circadian cycles. As a consequence, lungs were protected from inflammatory injury at times of day or in mouse mutants in which granule content was low. Changes in the proteome, granule content and NET formation also occurred in human neutrophils, and correlated with the incidence and severity of respiratory distress in pneumonia patients. Our findings unveil a 'disarming' strategy of neutrophils that depletes protein stores to reduce the magnitude of inflammation.

The combination of stable isotope labeling (SIL) with mass spectrometry (MS) allows comparison of the abundance of thousands of proteins in complex mixtures. However, interpretation of the large data sets generated by these techniques remains a challenge because appropriate statistical standards are lacking. Here, we present a generally applicable model that accurately explains the behavior of data obtained using current SIL approaches, including 18O, iTRAQ, and SILAC labeling, and different MS instruments. The model decomposes the total technical variance into the spectral, peptide, and protein variance components, and its general validity was demonstrated by confronting 48 experimental distributions against 18 different null hypotheses. In addition to its general applicability, the performance of the algorithm was at least similar than that of other existing methods. The model also provides a general framework to integrate quantitative and error information fully, allowing a comparative analysis of the results obtained from different SIL experiments. The model was applied to the global analysis of protein alterations induced by low H2O2 concentrations in yeast, demonstrating the increased statistical power that may be achieved by rigorous data integration. Our results highlight the importance of establishing an adequate and validated statistical framework for the analysis of high-throughput data.

MS-based quantitative proteomics plays an increasingly important role in biological and medical research and the development of these techniques remains one of the most important challenges in mass spectrometry. Numerous stable isotope labeling approaches have been proposed. However, and particularly in the case of 18 O-labeling, a standard protocol of general applicability is still lacking, and statistical issues associated to these methods remain to be investigated. In this work we present an improved high-throughput quantitative proteomics method based on whole proteome concentration by SDS-PAGE, optimized in-gel digestion, peptide 18 O-labeling, and separation by off-gel isoelectric focusing followed by liquid chromatography-LIT-MS. We demonstrate that the off-gel technique is fully compatible with 18 The analysis of differential protein expression is fundamental for the understanding of biological processes and plays an increasingly important role in biological and medical research (1). In recent years, numerous stable isotope labeling (SIL) 1 techniques have emerged as alternatives to the historically used two-dimensional-based approaches for semiquantitative proteomic studies. In these techniques the quantification is done in the same mass spectrometer where peptides are analyzed by tandem mass spectrometry (MS/MS), so relative quantification and peptide identification is performed at the same time. The differences among the several existing SIL approaches are mainly related to the way labels are introduced and the method used to perform the quantification by MS. Thus, in the SILAC method (2) labels are introduced metabolically at the protein level before peptides are generated from protein by enzymatic digestion, minimizing variability introduced by peptide preparation, whereas in the others labeling is performed postdigestion at the peptide level, either chemically in the iTRAQ method (3), or enzymatically in the 18 O labeling method (4 -6). In the iTRAQ method, quantification is made at the MS/MS level, allowing the possibility of performing multiplexed comparisons (7) whereas in SILAC and 18 O methods peptides are quantified at the MS level and are mainly used for pairwise comparisons. In other SIL approaches, such as the ICAT method (8), labeled peptides are specifically recovered after an affinity purification approach; this allows reducing peptide complexity, which is particularly appropriate to selectively analyze peptide subpopulations, such as reduced or oxidized cys-containing peptides (9). The 18 O labeling method has the advantage that labels are intro-

dAnaplasma phagocytophilum causes human granulocytic anaplasmosis. Infection with this zoonotic pathogen affects gene expression in both the vertebrate host and the tick vector, Ixodes scapularis. Here, we identified new genes, including spectrin alpha chain or alpha-fodrin (CG8) and voltage-dependent anion-selective channel or mitochondrial porin (T2), that are involved in A. phagocytophilum infection/multiplication and the tick cell response to infection. The pathogen downregulated the expression of CG8 in tick salivary glands and T2 in both the gut and salivary glands to inhibit apoptosis as a mechanism to subvert host cell defenses and increase infection. In the gut, the tick response to infection through CG8 upregulation was used by the pathogen to increase infection due to the cytoskeleton rearrangement that is required for pathogen infection. These results increase our understanding of the role of tick genes during A. phagocytophilum infection and multiplication and demonstrate that the pathogen uses similar strategies to establish infection in both vertebrate and invertebrate hosts.T icks are ectoparasites of animals and humans and are considered to be the most important arthropod vector of pathogens in some regions (1). Ixodes scapularis Say (Acari: Ixodidae) is an important vector of pathogens that infect and cause disease in humans and domestic animals in the United States. Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae), the focus of this study, is the causative agent of human, canine, and equine granulocytic anaplasmosis and tick-borne fever of ruminants (2, 3).A. phagocytophilum is an intracellular bacterium that infects vertebrate host neutrophils, where it multiplies within a parasitophorous vacuole, thus evading host defenses while inhibiting apoptosis and promoting cytoskeleton rearrangement for infection and multiplication (4-8). Tick-A. phagocytophilum interactions are not as well characterized as those between pathogen and vertebrate hosts (4). While A. phagocytophilum has been shown to infect I. scapularis gut cells (9) and salivary glands (10), the developmental cycle of this pathogen has not been described in ticks. Tick proteins such as Salp16, subolesin, antifreeze glycoprotein IAFGP, and alpha1-3-fucosyltransferease were differentially regulated and required for A. phagocytophilum infection of I. scapularis (10-20). Activation of heat shock proteins and other stress response proteins in ticks and cultured tick cells in response to A. phagocytophilum infection was also characterized by proteomics and transcriptomics analyses (21).The overall goal of our research is to characterize molecular interactions at the vector-pathogen interface and develop vaccines for the control of tick infestations and pathogen infection/transmission. Our hypothesis is that tick genes differentially expressed in response to pathogen infection would include those involved in pathogen infection, multiplication, and transmission, as well as in the tick protective response to infection. In this researc...

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Cardiovascular disease (CVD) is the leading cause of mortality in the world, with most CVD-related deaths resulting from myocardial infarction or stroke. The main underlying cause of thrombosis and cardiovascular events is atherosclerosis, an inflammatory disease that can remain asymptomatic for long periods. There is an urgent need for therapeutic and diagnostic options in this area. Atherosclerotic plaques contain autoantibodies 1,2 , and there is a connection between atherosclerosis and autoimmunity 3 . However, the immunogenic trigger and the effects of the autoantibody response during atherosclerosis are not well understood [3][4][5] . Here we performed high-throughput single-cell analysis of the atherosclerosis-associated antibody repertoire. Antibody gene sequencing of more than 1,700 B cells from atherogenic Ldlr −/− and control mice identified 56 antibodies expressed by in-vivo-expanded clones of B lymphocytes in the context of atherosclerosis. One-third of the expanded antibodies were reactive against atherosclerotic plaques, indicating that various antigens in the lesion can trigger antibody responses. Deep proteomics analysis identified ALDH4A1, a mitochondrial dehydrogenase involved in proline metabolism, as a target antigen of one of these autoantibodies, A12. ALDH4A1 distribution is altered during atherosclerosis, and circulating ALDH4A1 is increased in mice and humans with atherosclerosis, supporting the potential use of ALDH4A1 as a disease biomarker. Infusion of A12 antibodies into Ldlr −/− mice delayed plaque formation and reduced circulating free cholesterol and LDL, suggesting that anti-ALDH4A1 antibodies can protect against atherosclerosis progression and might have therapeutic potential in CVD.Atherosclerosis is a chronic inflammatory disease that leads to the formation of atheroma plaques in the arteries and is the main underlying cause of thrombosis, ischaemic heart disease and stroke 6 . The inflammatory reaction during atherosclerosis is believed to be triggered by the retention and subsequent oxidation (ox) of low-density lipoprotein (LDL) in the vessel sub-endothelium space 7 . In addition, the adaptive arm of the immune response is known to be critical during atherosclerosis [8][9][10] . Antibodies were first detected in atheroma plaques decades ago, and both protective and pathogenic functions have been attributed to B cells and the antibody immune response during atherosclerosis development 3,[11][12][13][14][15][16] . However, knowledge about the underlying antigenic triggers of this response and their effects on atherosclerosis 3 remains limited, as most studies have focused on oxidation-specific neoepitopes (OSEs) such as those contained in oxLDL [17][18][19][20][21][22] . Here, we performed an unbiased, high-throughput single-cell study of the antibody repertoire associated with atherosclerosis and identified an antibody-antigen pair with strong diagnostic and therapeutic potential.To study the antibody immune response associated with atherosclerosis, we made use of Ldlr −/− mi...