Single‐cell technologies are revolutionizing biology but are today mainly limited to imaging and deep sequencing. However, proteins are the main drivers of cellular function and in‐depth ...characterization of individual cells by mass spectrometry (MS)‐based proteomics would thus be highly valuable and complementary. Here, we develop a robust workflow combining miniaturized sample preparation, very low flow‐rate chromatography, and a novel trapped ion mobility mass spectrometer, resulting in a more than 10‐fold improved sensitivity. We precisely and robustly quantify proteomes and their changes in single, FACS‐isolated cells. Arresting cells at defined stages of the cell cycle by drug treatment retrieves expected key regulators. Furthermore, it highlights potential novel ones and allows cell phase prediction. Comparing the variability in more than 430 single‐cell proteomes to transcriptome data revealed a stable‐core proteome despite perturbation, while the transcriptome appears stochastic. Our technology can readily be applied to ultra‐high sensitivity analyses of tissue material, posttranslational modifications, and small molecule studies from small cell counts to gain unprecedented insights into cellular heterogeneity in health and disease.
Synopsis
A new ultra‐high sensitivity LC‐MS workflow increases sensitivity by up to two orders of magnitude and enables true single‐cell proteome analysis. In‐depth comparison indicates that the single‐cell transcriptome is stochastic while the single‐cell proteome is complete and stable.
A highly optimized data independent acquisition powered single‐cell proteomics workflow including sub‐µl sample preparation, very low flow chromatography and trapped ion mobility mass spectrometry (diaPASEF) is presented.
Single‐cell proteome analysis is performed by injecting cells one‐by‐one across the cell cycle into the LC‐MS and correctly identifies cell states.
Single‐cell proteome information is highly complementary to single‐cell transcriptome information.
At the single‐cell level the proteome is quantitatively and qualitatively stable, while the transcriptome is stochastic.
A new ultra‐high sensitivity LC‐MS workflow increases sensitivity by up to two orders of magnitude and enables true single‐cell proteome analysis. In‐depth comparison indicates that the single‐cell transcriptome is stochastic while the single‐cell proteome is complete and stable.
Although first described decades ago, the relevance of carbohydrate specific antibodies as mediators of type I allergy had not been recognized until recently. Previously, allergen specific IgE ...antibodies binding to carbohydrate epitopes were considered to demonstrate a clinically irrelevant cross-reactivity. However, this changed following the discovery of type I allergies specifically mediated by oligosaccharide structures. Especially the emerging understanding of red meat allergy characterized by IgE directed to the oligosaccharide alpha-gal showed that carbohydrate-mediated reactions can result in life threatening systemic anaphylaxis which in contrast to former assumptions proves a high clinical relevance of some carbohydrate allergens. Within the scope of this review article, we illustrate the historical development of carbohydrate-allergen-research, reaching from only diagnostically relevant crossreactive-carbohydrate-determinants to clinically important antigens mediating type I allergy. Focusing on clinical and immunological features of the alpha-gal syndrome, we highlight the discovery of oligosaccharides as potentially highly immunogenic antigens and mediators of type I allergy, report what is known about the route of sensitization and the immunological mechanisms involved in sensitization and elicitation phase of allergic responses as well as currently available diagnostic and therapeutic tools. Finally, we briefly report on carbohydrates being involved in type I allergies different from alpha-gal.
A versatile and facile synthetic route toward a ultralight hierarchical poroushybrid composed of metal‐organic gels and fluorinated graphene oxide is reported. The composite gels show excellent ...absorbency of oil and various organic solvents due to their prominent meso/macropores, notable hydrophobicity, and superoleophilicity.
To analyse predictors for relapses and number of attacks under different immunotherapies in patients with neuromyelitis optica spectrum disorder (NMOSD).
This is a retrospective cohort study ...conducted in neurology departments at 21 regional and university hospitals in Germany. Eligible participants were patients with aquaporin-4-antibody-positive or aquaporin-4-antibody-negative NMOSD. Main outcome measures were HRs from Cox proportional hazard regression models adjusted for centre effects, important prognostic factors and repeated treatment episodes.
265 treatment episodes with a mean duration of 442 days (total of 321 treatment years) in 144 patients (mean age at first attack: 40.9 years, 82.6% female, 86.1% aquaporin-4-antibody-positive) were analysed. 191 attacks occurred during any of the treatments (annual relapse rate=0.60). The most common treatments were rituximab (n=77, 111 patient-years), azathioprine (n=52, 68 patient-years), interferon-β (n=32, 61 patient-years), mitoxantrone (n=34, 32.1 patient-years) and glatiramer acetate (n=17, 10 patient-years). Azathioprine (HR=0.4, 95% CI 0.3 to 0.7, p=0.001) and rituximab (HR=0.6, 95% CI 0.4 to 1.0, p=0.034) reduced the attack risk compared with interferon-β, whereas mitoxantrone and glatiramer acetate did not. Patients who were aquaporin-4-antibody-positive had a higher risk of attacks (HR=2.5, 95% CI 1.3 to 5.1, p=0.009). Every decade of age was associated with a lower risk for attacks (HR=0.8, 95% CI 0.7 to 1.0, p=0.039). A previous attack under the same treatment tended to be predictive for further attacks (HR=1.5, 95% CI 1.0 to 2.4, p=0.065).
Age, antibody status and possibly previous attacks predict further attacks in patients treated for NMOSD. Azathioprine and rituximab are superior to interferon-β.
Purpose of Review
The alpha-Gal (α-Gal) syndrome is characterized by the presence of IgE antibodies directed at the carbohydrate galactose-alpha-1,3-galactose (α-Gal). In this article, we review the ...presence of α-Gal in food and non-food sources; we discuss the evolutionary context of the antibody response to α-Gal and highlight immune responses to α-Gal and other carbohydrates.
Recent findings
IgE antibodies have been associated with delayed allergy to red meat. In addition to food, drugs, and other products of animal origin are increasingly perceived as a risk for patients sensitized to α-Gal. The link between tick bites and anti-α-Gal IgE-antibody production that has been established first by epidemiological studies has now been confirmed in mouse models.
Summary
The anti-α-Gal immune response is complex and characterized by a unique feature. IgM and IgG antibodies have been found to confer protection against pathogens whereas the IgE-response to α-Gal is detrimental and causes severe reactions upon exposure to mammalian meat and other products.
The prevalence of food allergy is rising and is estimated to approach 10%. Red meat allergy is the first known food allergy elicited by immunoglobulin E (IgE) antibodies recognizing a carbohydrate. ...Due to the loss of function of the alpha-1,3-galactosyltransferase (
gene in humans, the disaccharide galactose-α-1,3-galactose (α-Gal) cannot be synthesized and therefore became immunogenic. IgE sensitization is elicited through the skin by repetitive tick bites transmitting α-Gal. The underlying mechanisms regarding innate and adaptive immune cell activation, including the B-cell isotype switch to IgE, are poorly understood, requiring further research and physiologically relevant animal models. Here, we describe a new animal model of red meat allergy using percutaneous α-Gal sensitization of gene-edited
-deficient pigs. Total and α-Gal-specific IgG, IgG1, IgG2, IgG4, and IgE levels were tracked. Further key factors associated with allergic skin inflammation, type 2 immunity, and allergy development were measured in PBMCs and skin samples. Significant increases in α-Gal-specific IgG1 and IgE levels indicated successful sensitization to the allergen α-Gal. Intracutaneous sensitizations with α-Gal recruited lymphocytes to the skin, including elevated numbers of T helper 2 (Th2) cells. Finally, α-Gal-sensitized pigs not only recognized α-Gal as non-self-antigen following α-Gal exposure through the skin but also developed anaphylaxis upon antigen challenge. Based on the similarities between the porcine and human skin, this new large animal model for α-Gal allergy should help to unveil the consecutive steps of cutaneous sensitization and aid the development of prophylactic and treatment interventions.
There is increasing evidence that C-reactive protein (CRP) can mediate inflammatory reactions following the transformation of functionally inert pentameric CRP (pCRP) into its structural isoform ...pCRP* and into monomeric CRP (mCRP). This conversion can occur on the membranes of apoptotic or activated cells or on extracellular vesicles (EVs) shed from the cell surface. Here, we characterized the association of CRP with EVs in plasma from sepsis patients using flow cytometry, and found highly elevated levels of total EV counts and CRP
EVs as compared to healthy individuals. We further assessed the ability of PentraSorb CRP, an extracorporeal device for the adsorption of CRP, to deplete free CRP and CRP
EVs. Treatment of septic plasma with the adsorbent in vitro resulted in almost complete removal of both, free CRP and CRP
EVs, while total EV counts remained largely unaffected, indicating the detachment of CRP from the EV surface. EVs from septic plasma elicited a release of interleukin-8 from cultured human monocytes, which was significantly reduced by adsorbent treatment prior to EV isolation. Our findings provide evidence that CRP
EVs exhibit pro-inflammatory characteristics and can contribute to the spreading of inflammation throughout the circulation on top of their pro-coagulant activity.
In biological fluids, proteins bind to the surface of nanoparticles to form a coating known as the protein corona, which can critically affect the interaction of the nanoparticles with living ...systems. As physiological systems are highly dynamic, it is important to obtain a time-resolved knowledge of protein-corona formation, development and biological relevancy. Here we show that label-free snapshot proteomics can be used to obtain quantitative time-resolved profiles of human plasma coronas formed on silica and polystyrene nanoparticles of various size and surface functionalization. Complex time- and nanoparticle-specific coronas, which comprise almost 300 different proteins, were found to form rapidly (<0.5 minutes) and, over time, to change significantly in terms of the amount of bound protein, but not in composition. Rapid corona formation is found to affect haemolysis, thrombocyte activation, nanoparticle uptake and endothelial cell death at an early exposure time.
There is the same number of n×n alternating sign matrices (ASMs) as there is of descending plane partitions (DPPs) with parts no greater than n, but finding an explicit bijection is an open problem ...for about 40 years now. So far, quadruples of statistics on ASMs and on DPPs that have the same joint distribution have been identified. We introduce extensions of ASMs and of DPPs along with n+3 statistics on each extension, and show that the two families of statistics have the same joint distribution. The ASM-DPP equinumerosity is obtained as an easy consequence by considering the (−1)-enumerations of these extended objects with respect to one pair of the n+3 pairs of statistics. One may speculate that the fact that these extensions might be necessary to have this significant increase in the number of statistics, as well as the involvement of signs when specializing to ASMs and DPPs may hint at the obstacles in finding an explicit bijection between ASMs and DPPs. One important tool for our proof is a multivariate generalization of the operator formula for the number of monotone triangles with prescribed bottom row that generalizes Schur functions.
Abstract
The direct response of the tropical mixed layer to near-inertial waves (NIWs) has only rarely been observed. Here, we present upper-ocean turbulence data that provide evidence for a strongly ...elevated vertical diffusive heat flux across the base of the mixed layer in the presence of a NIW, thereby cooling the mixed layer at a rate of 244 W m
−2
over the 20 h of continuous measurements. We investigate the seasonal cycle of strong NIW events and find that despite their local intermittent nature, they occur preferentially during boreal summer, presumably associated with the passage of atmospheric African Easterly Waves. We illustrate the impact of these rare but intense NIW induced mixing events on the mixed layer heat balance, highlight their contribution to the seasonal evolution of sea surface temperature, and discuss their potential impact on biological productivity in the tropical North Atlantic.
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