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Advances in several key technologies, including MHC peptidomics, have helped fuel our understanding of basic immune regulatory mechanisms and the identification of T cell receptor targets for the development of immunotherapeutics. Isolating and accurately quantifying MHC-bound peptides from cells and tissues enables characterization of dynamic changes in the ligandome due to cellular perturbations. However, the current multistep analytical process is challenging, and improvements in throughput and reproducibility would enable rapid characterization of multiple conditions in parallel. Here, we describe a robust and quantitative method whereby peptides derived from MHC-I complexes from a variety of cell lines, including challenging adherent lines such as MC38, can be enriched in a semiautomated fashion on reusable, dry-storage, customized antibody cartridges. Using this method, a researcher, with very little hands-on time and in a single day, can perform up to 96 simultaneous enrichments at a similar level of quality as a manual workflow. TOMAHAQ (Triggered by Offset, Multiplexed, Accurate-mass, High-resolution, and Absolute Quantification), a targeted mass spectrometry technique that combines sample multiplexing and high sensitivity, was employed to characterize neoepitopes displayed on MHC-I by tumor cells and to quantitatively assess the influence of neoantigen expression and induced degradation on neoepitope presentation. This unique combination of robust semiautomated MHC-I peptide isolation and high-throughput multiplexed targeted quantitation allows for both the routine analysis of >4000 unique MHC-I peptides from 250 million cells using nontargeted methods, as well as quantitative sensitivity down to the low amol/μl level using TOMAHAQ targeted MS., Competing Interests: Conflict of interest We are employed by Genentech, a member of the Roche group. The authors have no conflict of interest., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Tumor-specific mutations can generate neoantigens that drive CD8 T cell responses against cancer. Next-generation sequencing and computational methods have been successfully applied to identify mutations and predict neoantigens. However, only a small fraction of predicted neoantigens are immunogenic. Currently, predicted peptide binding affinity for MHC-I is often the major criterion for prioritizing neoantigens, although little progress has been made toward understanding the precise functional relationship between affinity and immunogenicity. We therefore systematically assessed the immunogenicity of peptides containing single amino acid mutations in mouse tumor models and divided them into two classes of immunogenic mutations. The first comprises mutations at a nonanchor residue, for which we find that the predicted absolute binding affinity is predictive of immunogenicity. The second involves mutations at an anchor residue; here, predicted relative affinity (compared with the WT counterpart) is a better predictor. Incorporating these features into an immunogenicity model significantly improves neoantigen ranking. Importantly, these properties of neoantigens are also predictive in human datasets, suggesting that they can be used to prioritize neoantigens for individualized neoantigen-specific immunotherapies., Competing Interests: Disclosures: Dr. Jhunjhunwala, Dr. Lupardus, and Dr. Delamarre reported a patent to US 20160069895 A1 pending. Dr. Wong reported personal fees from Oncomed Pharmaceuticals, personal fees from Array BioPharma, and personal fees from Pfizer outside the submitted work. Dr. de la Cruz reported "other" from Genentech outside the submitted work. Dr. Mellman reported personal fees from Genentech and grants from Genentech during the conduct of the study. No other disclosures were reported., (© 2020 Capietto et al.)

Human cells express thousands of different surface proteins that can be used for cell classification, or to distinguish healthy and disease conditions. A method capable of profiling a substantial fraction of the surface proteome simultaneously and inexpensively would enable more accurate and complete classification of cell states. We present a highly multiplexed and quantitative surface proteomic method using genetically barcoded antibodies called phage-antibody next-generation sequencing (PhaNGS). Using 144 preselected antibodies displayed on filamentous phage (Fab-phage) against 44 receptor targets, we assess changes in B cell surface proteins after the development of drug resistance in a patient with acute lymphoblastic leukemia (ALL) and in adaptation to oncogene expression in a Myc-inducible Burkitt lymphoma model. We further show PhaNGS can be applied at the single-cell level. Our results reveal that a common set of proteins including FLT3, NCR3LG1, and ROR1 dominate the response to similar oncogenic perturbations in B cells. Linking high-affinity, selective, genetically encoded binders to NGS enables direct and highly multiplexed protein detection, comparable to RNA-sequencing for mRNA. PhaNGS has the potential to profile a substantial fraction of the surface proteome simultaneously and inexpensively to enable more accurate and complete classification of cell states., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

ATP-dependent chromatin remodellers regulate access to genetic information by controlling nucleosome positions in vivo. However, the mechanism by which remodellers discriminate between different nucleosome substrates is poorly understood. Many chromatin remodelling proteins possess conserved protein domains that interact with nucleosomal features. Here we used a quantitative high-throughput approach, based on the use of a DNA-barcoded mononucleosome library, to profile the biochemical activity of human ISWI family remodellers in response to a diverse set of nucleosome modifications. We show that accessory (non-ATPase) subunits of ISWI remodellers can distinguish between differentially modified nucleosomes, directing remodelling activity towards specific nucleosome substrates according to their modification state. Unexpectedly, we show that the nucleosome acidic patch is necessary for maximum activity of all ISWI remodellers evaluated. This dependence also extends to CHD and SWI/SNF family remodellers, suggesting that the acidic patch may be generally required for chromatin remodelling. Critically, remodelling activity can be regulated by modifications neighbouring the acidic patch, signifying that it may act as a tunable interaction hotspot for ATP-dependent chromatin remodellers and, by extension, many other chromatin effectors that engage this region of the nucleosome surface.

Histone posttranslational modification leads to downstream effects indirectly by allowing or preventing docking of effector molecules, or directly by changing the intrinsic biophysical properties of local chromatin. To date, little has been done to study posttranslational modifications that lie outside of the unstructured tail domains of histones. Core residues, and in particular arginines in H3 and H4, mediate key interactions between the histone octamer and DNA in forming the nucleosomal particle. Using mass spectrometry, we find that one of these core residues, arginine 42 of histone H3 (H3R42), is dimethylated in mammalian cells by the methyltransferases coactivator arginine methyltransferase 1 (CARM1) and protein arginine methyltransferase 6 (PRMT6) in vitro and in vivo, and we demonstrate that methylation of H3R42 stimulates transcription in vitro from chromatinized templates. Thus, H3R42 is a new, "nontail" histone methylation site with positive effects on transcription. We propose that methylation of basic histone residues at the DNA interface may disrupt histone:DNA interactions, with effects on downstream processes, notably transcription.

The optimal orientation of the St. Jude Medical mechanical prosthesis in the mitral position has not yet been determined. While in the majority of cases the valve can perform satisfactorily regardless of valve orientation, certain circumstances can increase the risk of leaflet impingement. These valves are commonly implanted with their leaflets oriented parallel to the anatomic axis of the native leaflets (anatomic orientation) or with their prosthetic leaflets perpendicular to the axis of the native leaflets (antianatomic orientation). To determine the influence of valve orientation on the clearance from the prosthetic leaflet to the posterior ventricular wall, we calculated the clearances on all available models of the St. Jude Medical mitral valve. Clearances were computed from measurements of valve dimensions with use of an electronic caliper. In all cases the clearance in antianatomic orientation was at least 49.5% greater (mean 59%, range 49.5% to 77.5%) than in anatomic orientation.

In certain patients with anomalies of systemic venous connection, traditional transvenous pacemaker lead insertion may not be technically feasible. We report the use of the femoral venous approach to insert a permanent pacemaker in a patient with congenital heart disease who had undergone two previous cardiac operations and had persistent anomalies of the superior systemic venous circulation. We recommend that the femoral venous approach be considered in select patients requiring permanent pacing.

A 53-year-old man who had previously undergone resection of a left ventricular aneurysm was admitted because of hemoptysis. Preoperative evaluation with computed tomographic scan and cardiac catheterization demonstrated a pseudoaneurysm of the inferior ventricular wall measuring 16 cm in diameter with protrusion into the left hemithorax. The neck of the pseudoaneurysm was a defect in the ventricular wall extending from the base of the mitral valve annulus to the insertion of the posterior papillary muscle. Operative repair was performed using an albumin-coated, low-porosity Dacron patch.

The Björk-Shiley convexoconcave prosthetic valve has design characteristics that may result in a lower incidence of thromboembolic complications than the conventional spherical Björk-Shiley prosthesis. We evaluated the results of valve replacement with the convexoconcave prosthesis in 248 patients receiving 301 prosthetic valves between March, 1979, and June, 1981. One hundred thirteen patients had aortic valve replacement (AVR), 73 had mitral valve replacement (MVR), and 62 had multiple valve replacement. Two hundred nine (84%) were in New York Heart Association Class III or IV. The median duration of follow-up was 13 months, and follow-up information was available for 246 (99%) of the patients. The actuarial incidence of freedom from thromboembolism at two years was 98% in the AVR group, 97% in the MVR group, and 87% in the group having multiple valve replacement. There were no documented episodes of valve thrombosis or mechanical failure and no fatal thromboembolic complications. The absence of valve thrombosis is in marked contrast to the results reported with the spherical disc valve. Although longer follow-up is necessary, it appears that the convexoconcave design represents a major improvement in the Björk-Shiley prosthesis.