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Objective: The greatest challenge in the management of vulvar squamous cell carcinoma (VSCC) is treatment of recurrent disease where options for surgery and radiation have been exhausted, or treatment of disease where distant metastasis is present. Identification of mutations differentially expressed between tumor from patients who died of aggressive disease and tumor from patients with an indolent course could reveal novel prognostic indicators and guide development of therapeutic drugs., Methods: From 202 consecutive patients with VSCC, patients who recurred and died of disease (group A) were identified and matched by age, tumor size, depth of invasion and nodal status with those whose disease did not recur (group B). Tumors from 21 patients were subjected to whole exome sequencing of DNA and RNA, immunohistochemistry (IHC) antibodies of PD-L1 and P16, and in-situ hybridization (ISH) for high-risk HPV., Results: Analysis of DNA and RNA revealed six genes that were strongly differentially expressed between group A and B: TGM3, ACVR2A, ROS1, NFEL2, CCND1 and BCL6. Clinically relevant DNA mutations were significantly greater in group A versus B: 7 vs 2.3 mutations per patient. The most common genomic alterations were mutations in TP53 and the promoter region of TERT. Other common genomic events include alterations of FAT1, CDKN2A, PIK3CA, CCND1, and LRP1B. All samples were MSI stable and tumor mutational burden (TMB) was similar in groups A and B. Most VSCC specimens (81%) were positive for PD-L1., Conclusions: ACVR2A and TGM3 are significantly under-expressed in tumors with poor outcome, suggesting they may play a role in tumor suppression. Clinical outcome of VSCC appears independent of MSI, TMB, or PD-L1 status., Competing Interests: Declaration of Competing Interest All authors deny any competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

Identifying the protein targets of bioactive small molecules remains a major problem in the discovery of new chemical probes and therapeutics. While activity-based probes and photo-cross-linkers have had success in identifying protein targets of small molecules, each technique has limitations. Here we describe a method for direct proximity tagging of proteins that bind small molecules. We engineered a promiscuous ligase based on the NEDD8 conjugating enzyme, Ubc12, which can be covalently linked to a small molecule of interest. When target proteins bind the small molecule, they are directly labeled on surface lysines with a biotinylated derivative of the small ubiquitin homologue, NEDD8. This unique covalent tag can then be used to identify the small molecule binding proteins. Utilizing the drug dasatinib, we have shown that dasatinib-directed NEDDylation occurs for known endogenous protein binders in complex cell lysates. In addition, we have been able to improve NEDDylation efficiency through rational mutagenesis. Finally, we have shown that affinity-directed NEDDylation can be applied to two other protein-ligand interactions beyond kinases. Proximity tagging using this engineered ligase requires direct binding of the target and, thus, provides a useful and orthogonal approach to facilitate small molecule target identification., Competing Interests: Notes The authors declare no competing financial interest.

Development of tool molecules that inhibit Jumonji demethylases allows for the investigation of cancer-associated transcription. While scaffolds such as 2,4-pyridinedicarboxylic acid (2,4-PDCA) are potent inhibitors, they exhibit limited selectivity. To discover new inhibitors for the KDM4 demethylases, enzymes overexpressed in several cancers, we docked a library of 600,000 fragments into the high-resolution structure of KDM4A. Among the most interesting chemotypes were the 5-aminosalicylates, which docked in two distinct but overlapping orientations. Docking poses informed the design of covalently linked fragment compounds, which were further derivatized. This combined approach improved affinity by ∼ 3 log-orders to yield compound 35 (Ki = 43 nM). Several hybrid inhibitors were selective for KDM4C over the related enzymes FIH, KDM2A, and KDM6B while lacking selectivity against the KDM3 and KDM5 subfamilies. Cocrystal structures corroborated the docking predictions. This study extends the use of structure-based docking from fragment discovery to fragment linking optimization, yielding novel KDM4 inhibitors., Competing Interests: Notes The authors declare no competing financial interest.

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 low reactivity of peptide-prolyl-thioesters in native chemical ligation is not due to steric effects at the β-carbon, but rather to the presence of a carbonyl moiety on the nitrogen atom of the proline.

This review describes the techniques currently used for quantitative neurophysiologic measurement during cardiac surgery and their potential impact on clinical outcome. Electroencephalography (EEG) characterizes cerebrocortical neuronal electrical activity and was part of some of the earliest cardiopulmonary bypass procedures, yet today it is not widespread use. Each of the common misunderstandings regarding a supposed limitation of this technology is explained. Its major genuine shortcoming, a lack of selectivity, may now be overcome with the combined use of additional monitoring modalities. The influence of intracranial hemodynamics on observed EEG changes may be determined continuously and noninvasively with transcranial Doppler (TCD) ultrasound. TCD provides an indication of sudden change in either blood flow or vascular resistance as well as the detection of emboli. In addition, the metabolic status of cortical neurons can be monitored by regional cerebral venous oxygen saturation (rCVOS) using noninvasive transcranial near-infrared spectroscopy. The % rCVOS tends to remain remarkably stable over a wide range of temperatures, perfusion pressures, and anesthetic states. Marked change in either direction signifies a serious imbalance between oxygen delivery and consumption. Measurement of rCVOS does not require blood flow, pulsatile or otherwise, so that it offers the only means of monitoring during circulatory arrest. By characterizing the dynamic interplay among cerebral hemodynamics, metabolism, and electrogenesis, these technologies permit the rapid detection and correction of potentially hazardous conditions.

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.

Percutaneous cardiopulmonary bypass (PCPB) has recently come to the forefront of medicine as a technique for resuscitating and supporting patients in various clinical situations. Current systems utilize small-diameter cannulas to aspirate blood under high suction into the cardiopulmonary bypass circuit. Aspiration-based systems have several disadvantages including risk of air embolism, blood hemolysis, and cavitation. Additionally, they are suboptimal for use during open-heart surgical procedures. A system with a venous cannula that employs gravity drainage has been evaluated. Once advanced into position over a guide-wire, the stylet is removed, causing the basket near the end of the cannula to expand. Blood flows into the cannula from side holes and the basket region, which prevents the vessel wall or atrium from collapsing around the catheter and impeding venous drainage. Hemodynamic, hematologic, and histologic examinations were performed on eight anesthetized mongrel dogs during 2 h of PCPB. All animals exhibited adequate tissue perfusion and right and left heart decompression. All animals were successfully weaned from PCPB and after 30 min exhibited normal myocardial function. No ischemic changes were observed in the heart, lung, kidney, or liver by light and electron microscopy. We conclude that full PCPB can be satisfactorily achieved by using a novel percutaneous venous cannula and gravity drainage.

Nineteen intra-aortic balloon (IAB) ruptures occurred in sixteen patients during a three-year period. Perforation occurred secondary to abrasion with material failure or mishandling of the device during insertion. To avoid serious sequelae, it is important to be aware of the possibility of IAB rupture and to remove any defective device immediately upon recognition of an event.

Early revascularization is critical for the treatment of acute myocardial infarction (AMI) because the ischemic myocardium begins to suffer irreversible damage after 4 h from the onset of symptoms. However, to make a diagnosis, perform coronary angiography, and prepare for operative revascularization usually takes longer than 4 h. Also, once a patient develops severe cardiogenic shock, coronary angiography is often impossible. Without angiography, the patient is no longer a candidate for surgical repair. To circumvent this problem, we designed this experiment to determine whether intraoperative aortic root angiography after cardiopulmonary bypass and cardioplegic arrest could satisfactorily substitute for angiographic examination in the identification of critical coronary lesions. The feasibility of this approach was tested in dog hearts in which one or tow of the major coronary arteries were ligated. The ascending aorta was then clamped, contrast material was injected, and continuous real-time fluoroscopic images were obtained and recorded on videotape. The videotape was then analyzed by three physicians independently, each without prior knowledge of the lesion locations. Lesions of the left anterior descending artery, the circumflex coronary artery, and the right coronary artery were identified with 94, 91, and 94% accuracy, respectively, for an overall identification rate of 92%. We conclude that aortic root angiography reliably demonstrates coronary artery lesions, and refinements in this technique may allow certain patients to undergo coronary operations without preoperative catheterization.

From January 1973 to December 1989, 54 patients over 80 years of age underwent an intracardiac repair which included a cardiac valve operation. There were 21 males and 33 females. Mean age at operation was 82 years, range 80-89 years. Fifty-two patients (96%) were in New York Heart Association functional class III or IV preoperatively. Six patients had undergone previous valve surgery (11%). There were eight hospital deaths (14.8%). Risk factors for hospital death included older age at repair (p = 0.008), increased total cardiopulmonary bypass time (p = 0.06), and, possibly, smaller aortic valve prosthesis (p = 0.10). All 46 hospital survivors were followed up at a mean of 28.8 months after hospital discharge. There were 11 late deaths (23.9%), occurring at a mean of 32.3 months postoperatively. Survival analysis indicated that increased age (p = 0.06) and increased pulmonary artery diastolic pressure preoperatively (p less than 0.07) were multivariate risk factors for overall mortality. Actuarial survival at 5 years was 44%, with no difference from survival in an age-, sex-, and race-matched population. We conclude that octogenarians in the modern era have good chance for survival following valvular surgery. As hazards for full anticoagulation were low in this series, if valve repair is not feasible, we presently recommend the use of mechanical valves in the elderly to reduce the requirement for late reoperation due to bioprosthesis degeneration.

Spinal cord hypoperfusion injury is a devastating complication of cross-clamping the proximal thoracic aorta. The collateral circulation around the cross-clamp is generally poorly developed, and the run-off is immense, resulting in extremely low thoracic aortic and spinal cord perfusion pressures. The authors postulated that balloon occlusion of the abdominal aorta might confine this reduced collateral flow around the cross-clamp to the thoracic aorta. In 8 of 16 dogs subjected to aortic cross-clamping of the aorta just beyond the arch vessels, the abdominal aorta was also occluded by a balloon. Thoracic aortic pressure and spinal cord perfusion pressure were significantly higher in the animals with aortic balloon occlusion than in those without balloon occlusion (77 +/- 8 mm Hg versus 26 +/- 1 mm Hg, p less than 0.01, and 67 +/- 8 mm Hg versus 18 +/- 2 mm Hg, p less than 0.01, at 10 minutes after cross-clamping). Abdominal aortic balloon occlusion increases thoracic aortic pressure after the aorta is cross-clamped proximally. Further studies are necessary in primates to assess the effect of this procedure in spinal cord perfusion and the rate of paraplegia.

From March 1978 through July 1985, 23 patients underwent implantation of 24 intraluminal ringed prostheses (IRP). There were 18 men and 5 women, with a mean age of 54.7 years, range 15-74 years. Eleven IRP were placed in the ascending aorta, two in the transverse arch, and 11 in the descending aorta. Pathology included acute aortic dissection in four patients, chronic dissection in four, and aortic aneurysm in 16. There were eight hospital deaths (35%). Causes of death included acute cardiac failure in seven patients, and ruptured abdominal aortic aneurysm in one. IRP complications requiring revision included right coronary artery occlusion in three of 11 patients (27%) with an IRP in the ascending aorta. Graft revision was also required in three of 11 IRP implanted in the descending aorta (27%), due to graft occlusion in one and graft stenosis in two. Of the six patients with IRP complications, there were three hospital deaths (50%). All 15 hospital survivors were followed for a mean of 68.5 months, range 5-112 months. There were four late deaths (26.7%). Causes of late death included hemoptysis in one, cardiomyopathy in one, and aortic redissection and rupture in two. We conclude that patients undergoing repair of aortic pathology with IRP have an important risk of early phase events, as technical problems can occur due to malposition and slippage of the securing rings.

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.

Fifty-five patients underwent amputation of the leg. Fifty-eight percent of the amputations were above-knee and 32 percent below-knee. Preoperative Doppler ankle blood pressure measurements and ankle/brachial ratios were compared and correlated with wound healing in patients with below-knee amputation. Statistical analysis documented that such measurements were significant in predicting wound healing. If blood pressure greater than 55 mm Hg at the knee, greater than 70 mm Hg at the incision site or greater than 70 mm Hg at the ankle or an ankle/brachial ratio of at least 0.3 is documented, satisfactory healing will follow amputation.