Treatment of Symptomatic Carotid Webs: A Single Center Review.

INTRODUCTION: Carotid webs are abnormal thin shelf-like or flap-like tissue in the carotid bulb (proximal internal carotid artery). Rarely are carotid webs detected prior to symptoms since routine carotid artery surveillance is not performed in younger individuals without traditional risk factors for carotid disease. The cause and natural history remain unknown. In general, they are not common, but should be considered in the differential diagnosis of a patient who presents with ischemic neurologic symptoms. The web can create a flow disturbance, potentiating local thrombus formation, which can embolize producing resulting in cerebral ischemia. Current treatment is to reduce thrombus formation (antithrombotics and/or anticoagulation) or to alter the flow disturbance caused by the web (surgical removal or stent). METHODS: We retrospectively identified all patients presenting with acute ischemic stroke to our Comprehensive Stroke Center that were diagnosed with carotid web from January 2020 to December 2023. Patient demographics, presentation, hospital course including treatment and complications were collected and reported. RESULTS: Fifteen patients presented with carotid web and stroke from 2020-2023 and 13 underwent carotid artery stenting or endarterectomy with no periprocedural complications. Most (40%) carotid webs were not primarily identified by the initial radiology interpretation. DISCUSSION: We discuss our experience of carotid web and its management as well as review of the current literature.

Digital health's influence on the association between birth preference and vaginal birth.

BACKGROUND: Women's preferred mode of birth during pregnancy is predictive of their actual mode of birth. Digital prenatal care services are a promising method for educating women on mode of birth to reduce elective cesareans. This study aimed to evaluate the influence of digital health on the association between birth preference and mode of birth. METHODS: Data come from 5409 pregnant women enrolled in a digital platform for women's and family health. Multi-trajectory modeling identified trajectories of digital health usage throughout pregnancy. Adjusted logistic regression models tested associations between birth preferences and mode of birth. The modifying effect of digital health usage on the association between birth preference and mode of birth was assessed on the multiplicative scale. RESULTS: Four distinct trajectories of digital service usage were identified and labeled as: (1) baseline users (52%): the reference group; (2) just-in timers (16%): high usage during the third trimester; (3) learners (26%): high educational resource usage (e.g., articles and classes) throughout pregnancy; and (4) super users (6%): high usage of both education and care resources throughout pregnancy. Overall, preferred mode of birth at enrollment was predictive of actual mode of birth; however, digital health usage moderated this association, whereby super users and learners who preferred a cesarean at enrollment were more likely to deliver vaginally, compared to baseline users who preferred a cesarean. CONCLUSION: For the increasing proportion of women considering an elective cesarean, education through a prenatal digital health platform may help to encourage vaginal birth and reduce cesarean births.

Reclaiming narratives of empowerment around Black maternal health: a strengths-based, community-informed focus group study.

OBJECTIVES: Research on Black maternal populations often focuses on deficits that can reinforce biases against Black individuals and communities. The research landscape must shift towards a strengths-based approach focused on the protective assets of Black individuals and communities to counteract bias. This study engaged the local Black community using a strengths-based approach to discuss the assets of Black maternal populations and to inform the design of a future clinical trial focused on reducing Black maternal health disparities. DESIGN: Guided by the Theory of Maternal Adaptive Capacity, we conducted three purposive focus group sessions with Black adult community members. The focus groups were semi-structured to cover specific topics, including the strengths of the local community, strengths specific to pregnant community members, how the strengths of community members can support pregnant individuals, and how the strengths of pregnant community members can facilitate a healthy pregnancy. The focus group interviews were transcribed verbatim and analyzed using thematic content analysis. RESULTS: Three focus group sessions were conducted with sixteen female individuals identifying as Black or African American. Central themes include (1) the power of pregnancy and motherhood in Black women, (2) challenging negative perceptions and media representation of Black mothers, (3) recognizing history and reclaiming cultural traditions surrounding birth, and (4) community as the foundation of Black motherhood. CONCLUSION: Black community members identified powerful themes on Black maternal health through a strengths-based lens. These focus groups fostered relationships with the Black community, elucidated possible solutions to improve Black women's health and wellness, and offered direction on our research design and intervention.

Neurologic Statistical Prognostication and Risk Assessment for Kids on Extracorporeal Membrane Oxygenation-Neuro SPARK.

This study presents Neuro-SPARK, the first scoring system developed to assess the risk of neurologic injury in pediatric and neonatal patients on extracorporeal membrane oxygenation (ECMO). Using the extracorporeal life support organization (ELSO) registry, we applied robust machine learning methodologies and clinical expertise to a 10 years dataset. We produced separate models for veno-venous (V-V ECMO) and veno-arterial (V-A ECMO) configurations due to their different risk factors and prevalence of neurologic injury. Our models identified 14 predictor variables for V-V ECMO and 20 for V-A ECMO, which demonstrated moderate accuracy in predicting neurologic injury as defined by the area under the receiver operating characteristic (AUROC) (V-V = 0.63, V-A = 0.64) and good calibration as measured by the Brier score (V-V = 0.1, V-A = 0.15). Furthermore, our post-hoc analysis identified high- and low-risk groups that may aid clinicians in targeted neuromonitoring and guide future research on ECMO-associated neurologic injury. Despite the inherent limitations, Neuro-SPARK lays the foundation for a risk-assessment tool for neurologic injury in ECMO patients, with potential implications for improved patient outcomes.

Nanosecond solvation dynamics in a polymer electrolyte for lithium batteries.

Solvation dynamics critically affect charge transport. Spectroscopic experiments and computer simulations show that these dynamics in aqueous systems occur on a picosecond timescale. In the case of organic electrolytes, however, conflicting values ranging from 1 to several 100 picoseconds have been reported. We resolve this conflict by studying mixtures of an organic polymer and a lithium salt. Lithium ions coordinate with multiple polymer chains, resulting in temporary crosslinks. Relaxation of these crosslinks, detected by quasielastic neutron scattering, are directly related to solvation dynamics. Simulations reveal a broad spectrum of relaxation times. The average timescale for solvation dynamics in both experiment and simulation is one nanosecond. We present the direct measurement of ultraslow dynamics of solvation shell break-up in an electrolyte.

Photocatalytic Activation of Aryl(trifluoromethyl) Diazos to Carbenes for High-Resolution Protein Labeling with Red Light.

State-of-the-art methods in photoproximity labeling center on the targeted generation and capture of short-lived reactive intermediates to provide a snapshot of local protein environments. Diazirines are the current gold standard for high-resolution proximity labeling, generating short-lived aryl(trifluoromethyl) carbenes. Here, we present a method to access aryl(trifluoromethyl) carbenes from a stable diazo source via tissue-penetrable, deep red to near-infrared light (600-800 nm). The operative mechanism of this activation involves Dexter energy transfer from photoexcited osmium(II) photocatalysts to the diazo, thus revealing an aryl(trifluoromethyl) carbene. The labeling preferences of the diazo probe with amino acids are studied, showing high reactivity toward heteroatom-H bonds. Upon the synthesis of a biotinylated diazo probe, labeling studies are conducted on native proteins as well as proteins conjugated to the Os photocatalyst. Finally, we demonstrate that the conjugation of a protein inhibitor to the photocatalyst also enables selective protein labeling in the presence of spectator proteins and achieves specific labeling of a membrane protein on the surface of mammalian cells via a two-antibody photocatalytic system.

The pathogenic T42A mutation in SHP2 rewires the interaction specificity of its N-terminal regulatory domain.

Mutations in the tyrosine phosphatase SHP2 are associated with a variety of human diseases. Most mutations in SHP2 increase its basal catalytic activity by disrupting auto-inhibitory interactions between its phosphatase domain and N-terminal SH2 (phosphotyrosine recognition) domain. By contrast, some disease-associated mutations located in the ligand-binding pockets of the N- or C-terminal SH2 domains do not increase basal activity and likely exert their pathogenicity through alternative mechanisms. We lack a molecular understanding of how these SH2 mutations impact SHP2 structure, activity, and signaling. Here, we characterize five SHP2 SH2 domain ligand-binding pocket mutants through a combination of high-throughput biochemical screens, biophysical and biochemical measurements, and molecular dynamics simulations. We show that, while some of these mutations alter binding affinity to phosphorylation sites, the T42A mutation in the N-SH2 domain is unique in that it also substantially alters ligand-binding specificity, despite being 8-10 Å from the specificity-determining region of the SH2 domain. This mutation exerts its effect on sequence specificity by remodeling the phosphotyrosine binding pocket, altering the mode of engagement of both the phosphotyrosine and surrounding residues on the ligand. The functional consequence of this altered specificity is that the T42A mutant has biased sensitivity toward a subset of activating ligands and enhances downstream signaling. Our study highlights an example of a nuanced mechanism of action for a disease-associated mutation, characterized by a change in protein-protein interaction specificity that alters enzyme activation.

Association Between Doula Use on a Digital Health Platform and Birth Outcomes.

OBJECTIVE: To examine the association between the use of virtual doula appointments on a comprehensive digital health platform and users' mode of birth and their birth experiences, among all platform users and Black platform users. METHODS: Data for this retrospective cohort study were extracted from individuals who enrolled in a comprehensive digital health platform, between January 1, 2020, and April 22, 2023. Multivariable logistic regression models were used to estimate the association between number of virtual doula appointments completed on the digital health platform and odds of cesarean birth and user-reported birth experience outcomes, which included help deciding a birth preference, receiving a high level of support during pregnancy, learning medically accurate information about pregnancy complications and warning signs, and managing mental health during pregnancy, stratified by parity. The interaction of doula utilization by race for each outcome was also tested. RESULTS: Overall 8,989 platform users were included. The completion of at least two appointments with a virtual doula on the digital health platform was associated with a reduction in odds of cesarean birth among all users (adjusted odds ratio [aOR] 0.80, 95% CI, 0.65-0.99) and among Black users (aOR 0.32, 95% CI, 0.14-0.72). Among platform users with a history of cesarean birth, completion of any number of doula visits was associated with a reduction in odds of repeat cesarean birth (one visit: aOR 0.35, 95% CI, 0.17-0.72; two or more visits: aOR 0.37, 95% CI, 0.17-0.83). Analyses among all users indicated dose-response associations between increased virtual doula use and greater odds of users reporting support in deciding a birth preference (one visit: aOR 2.35, 95% CI, 2.02-2.74; two or more visits: aOR 3.67, 95% CI, 3.03-4.44), receiving a high level of emotional support during pregnancy (one visit: aOR 1.99, 95% CI, 1.74-2.28; two or more visits: aOR 3.26, 95% CI, 2.70-3.94), learning medically accurate information about pregnancy complications and warning signs (one visit: aOR 1.26, 95% CI, 1.10-1.44; two or more visits: aOR 1.55, 95% CI, 1.29-1.88), and help managing mental health during pregnancy (one visit: aOR 1.28, 95% CI, 1.05-1.56; two or more visits: aOR 1.78, 95% CI, 1.40-2.26). CONCLUSION: This analysis demonstrates that virtual doula support on a digital health platform is associated with lower odds of cesarean birth and an improved birth experience. Positive findings among Black users and users with vaginal birth after cesarean suggest that doula support is critical for patient advocacy, and that digital health may play a meaningful role in increasing health equity in birth outcomes.

TCR signaling promotes formation of an STS1-Cbl-b complex with pH-sensitive phosphatase activity that suppresses T cell function in acidic environments.

T cell responses are inhibited by acidic environments. T cell receptor (TCR)-induced protein phosphorylation is negatively regulated by dephosphorylation and/or ubiquitination, but the mechanisms underlying sensitivity to acidic environments are not fully understood. Here, we found that TCR stimulation induced a molecular complex of Cbl-b, an E3-ubiquitin ligase, with STS1, a pH-sensitive unconventional phosphatase. The induced interaction depended upon a proline motif in Cbl-b interacting with the STS1 SH3 domain. STS1 dephosphorylated Cbl-b interacting phosphoproteins. The deficiency of STS1 or Cbl-b diminished the sensitivity of T cell responses to the inhibitory effects of acid in an autocrine or paracrine manner in vitro or in vivo. Moreover, the deficiency of STS1 or Cbl-b promoted T cell proliferative and differentiation activities in vivo and inhibited tumor growth, prolonged survival, and improved T cell fitness in tumor models. Thus, a TCR-induced STS1-Cbl-b complex senses intra- or extra-cellular acidity and regulates T cell responses, presenting a potential therapeutic target for improving anti-tumor immunity.

Role of cardiopulmonary interactions in development of ventilator-induced lung injury-Experimental evidence and clinical Implications.

Ventilator-induced lung injury (VILI) impacts outcomes in ARDS and optimization of ventilatory strategies improves survival. Decades of research has identified various mechanisms of VILI, largely focusing on airspace forces of plateau pressure, tidal volume and driving pressure. Experimental evidence indicates the role of adverse cardiopulmonary interaction during mechanical ventilation, contributing to VILI genesis mostly by modulating pulmonary vascular dynamics. Under passive mechanical ventilation, high transpulmonary pressure increases afterload on right heart while high pleural pressure reduces the RV preload. Together, they can result in swings of pulmonary vascular flow and pressure. Altered vascular flow and pressure result in increased vascular shearing and wall tension, in turn causing direct microvascular injury accompanied with permeability to water, proteins and cells. Moreover, abrupt decreases in airway pressure, may result in sudden overperfusion of the lung and result in similar microvascular injury, especially when the endothelium is stretched or primed at high positive end-expiratory pressure. Microvascular injury is universal in VILI models and presumed in the diagnosis of ARDS; preventing such microvascular injury can reduce VILI and impact outcomes in ARDS. Consequently, developing cardiovascular targets to reduce macro and microvascular stressors in the pulmonary circulation can potentially reduce VILI. This paper reviews the role of cardiopulmonary interaction in VILI genesis.

Chimney-Shaped Phase Diagram in a Polymer Blend Electrolyte.

The phase behavior of polymer blend electrolytes comprising poly(ethylene oxide) (PEO)/poly(methyl methacrylate) (PMMA)/lithium bis(trifluoromethanesulfonyl)imide (LiTFSI) was determined using a combination of light and small angle neutron scattering (SANS) experiments. The results at a fixed temperature (110 °C) are presented on a PEO concentration versus salt (LiTFSI) concentration plot. The blends are miscible at all PEO concentrations in the absence of salt. With added salt, a region of immiscibility is obtained in PEO-lean polymer blend electrolytes; blends rich in PEO remain miscible at most salt concentrations. A narrow region of immiscibility juts into the miscible region, giving the phase diagram a chimney-like appearance. The data are qualitatively consistent with a simple extension of Flory-Huggins theory with a composition-dependent Flory-Huggins interaction parameter, χ, that was determined independently from SANS data from homogeneous blend electrolytes. Phase diagrams like the one we obtained were anticipated by self-consistent field theory calculations that account for correlations between ions. The relationship between these theories and measured χ remains to be established.

Association Between the Use of a Digital Health Platform During Pregnancy and Helping Users Avoid Emergency and In-Person Care: Retrospective Observational Study.

BACKGROUND: Almost one-third of pregnant people visit the emergency room during pregnancy. Although some emergency care is necessary, gaps in patient education and inaccessibility of preventive services have been identified as key reasons for high-cost, low-value care in pregnancy. Digital platforms present a promising solution for providing resources to supplement routine prenatal care, thereby reducing the use of low-value in-person services. OBJECTIVE: This study aimed to describe the relationship between the use of Maven and in-person care avoidance (emergency room or office visits) during pregnancy. Maven is a digital prenatal health platform that supplements routine prenatal care. Maven offers educational content (articles, videos, and classes), care coordination (through a care advocate), and provider services (web-based appointments and communication with providers) designed to complement prenatal care. Specifically, the aims of this study were to examine whether the use of Maven is associated with in-person care avoidance overall and whether improvements in pregnancy-related knowledge facilitate in-person care avoidance. To assess aim 2, we tested if the use of Maven is associated with improvements in self-reported understanding of warning signs and medically accurate information and if self-reported understanding of medically accurate information and warning signs is associated with in-person care avoidance in a population of Maven users. METHODS: In this retrospective study, we used adjusted logistic regression to examine the relationship between digital platform use, avoidance of in-person care, and the platform's influence on pregnancy-related knowledge (learning medically accurate information and recognizing warning signs). Demographics, medical history, and in-person care avoidance were self-reported. RESULTS: Of the 5263 users, 280 (5.32%) reported that Maven helped them avoid in-person care during pregnancy. More users who reported avoiding in-person care also reported that the digital platform helped them understand warning signs (231/280, 82.5%) and learned medically accurate information (185/280, 66.1%). In the adjusted models, all modes of digital service use (assessed as quartiles) were associated with avoiding in-person care in a dose-response manner (eg, web-based provider appointments: Q2 adjusted odds ratio [aOR] 1.57, 95% CI 1.00-2.41; Q3 aOR 2.53, 95% CI 1.72-3.72; Q4 aOR 5.26, 95% CI 3.76-7.42). Users were more likely to avoid in-person care if they reported that Maven helped them recognize warning signs (aOR 3.55, 95% CI 2.60-4.94) or learn medically accurate information (aOR 2.05, 95% CI 1.59-2.67). CONCLUSIONS: These results suggest that digital platforms can be effective in helping patients to avoid in-person care. The educational pathway suggests that digital platforms can be particularly effective in helping patients recognize warning signs and learn medically accurate information, which may help them avoid in-person care by recognizing when in-person care is medically appropriate. Future work is needed to assess other pathways through which digital resources can support pregnant people and improve perinatal care use.

Utilization of digital prenatal services and management of depression and anxiety during pregnancy: A retrospective observational study.

Introduction: We examined how utilization of Maven, a digital healthcare platform that provides virtual prenatal services, is associated with improvements in perceived management of anxiety and depression during pregnancy, and how medical knowledge and support may influence this association. Materials and Methods: In this retrospective study we used adjusted logistic regression to examine the relationship between digital platform use in pregnancy and perceived mental health management, and how perceived management of mental health is affected by user-reported improvements in medical knowledge and feeling supported by the platform. Effects were evaluated separately among users with and without a mental health condition. Demographics, medical history, and mental health management were self-reported. Results: Of 5,659 users, 705 (12.5%) reported that Maven helped them manage anxiety and/or depression in the prenatal period. In adjusted models, users who read more articles, sent more messages to care advocates, or had more appointments with providers were more likely to report improved management of mental health in a dose-response manner (e.g., articles read: Q2 aOR 1.31 (95% CI 1.01-1.70), Q3 aOR 1.68 (95% CI 1.30-2.17), Q4 1.99 (95% CI 1.54-2.59)). Improvements in medical knowledge and high perceived support were both associated with better perceived mental health management. Results were similar in users with and without a mental health condition. Discussion: These results suggest that access to a diverse set of digital resources provides multiple pathways to managing depression and anxiety during pregnancy for those with and without a diagnosed mental health condition.

Validation of extracorporeal membrane oxygenation mortality prediction and severity of illness scores in an international COVID-19 cohort.

BACKGROUND: Veno-venous extracorporeal membrane oxygenation (V-V ECMO) is a lifesaving support modality for severe respiratory failure, but its resource-intensive nature led to significant controversy surrounding its use during the COVID-19 pandemic. We report the performance of several ECMO mortality prediction and severity of illness scores at discriminating survival in a large COVID-19 V-V ECMO cohort. METHODS: We validated ECMOnet, PRESET (PREdiction of Survival on ECMO Therapy-Score), Roch, SOFA (Sequential Organ Failure Assessment), APACHE II (acute physiology and chronic health evaluation), 4C (Coronavirus Clinical Characterisation Consortium), and CURB-65 (Confusion, Urea nitrogen, Respiratory Rate, Blood Pressure, age >65 years) scores on the ISARIC (International Severe Acute Respiratory and emerging Infection Consortium) database. We report discrimination via Area Under the Receiver Operative Curve (AUROC) and Area under the Precision Recall Curve (AURPC) and calibration via Brier score. RESULTS: We included 1147 patients and scores were calculated on patients with sufficient variables. ECMO mortality scores had AUROC (0.58-0.62), AUPRC (0.62-0.74), and Brier score (0.286-0.303). Roch score had the highest accuracy (AUROC 0.62), precision (AUPRC 0.74) yet worst calibration (Brier score of 0.3) despite being calculated on the fewest patients (144). Severity of illness scores had AUROC (0.52-0.57), AURPC (0.59-0.64), and Brier Score (0.265-0.471). APACHE II had the highest accuracy (AUROC 0.58), precision (AUPRC 0.64), and best calibration (Brier score 0.26). CONCLUSION: Within a large international multicenter COVID-19 cohort, the evaluated ECMO mortality prediction and severity of illness scores demonstrated inconsistent discrimination and calibration highlighting the need for better clinically applicable decision support tools.

Early Changes in Arterial Partial Pressure of Carbon Dioxide and Blood Pressure After Starting Extracorporeal Membrane Oxygenation in Children: Extracorporeal Life Support Organization Database Study of Neurologic Complications.

OBJECTIVE: Neurologic complications in pediatric patients supported by extracorporeal membrane oxygenation (ECMO) are common and lead to morbidity and mortality; however, few modifiable factors are known. DESIGN: Retrospective study of the Extracorporeal Life Support Organization registry (2010-2019). SETTING: Multicenter international database. PATIENTS: Pediatric patients receiving ECMO (2010-2019) for all indications and any mode of support. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We investigated if early relative change in Pa co2 or mean arterial blood pressure (MAP) soon after starting ECMO was associated with neurologic complications. The primary outcome of neurologic complications was defined as a report of seizures, central nervous system infarction or hemorrhage, or brain death. All-cause mortality (including brain death) was used as a secondary outcome.Out of 7,270 patients, 15.6% had neurologic complications. Neurologic complications increased when the relative Pa co2 decreased by greater than 50% (18.4%) or 30-50% (16.5%) versus those who had a minimal change (13.9%, p < 0.01 and p = 0.046). When the relative MAP increased greater than 50%, the rate of neurologic complications was 16.9% versus 13.1% those with minimal change ( p = 0.007). In a multivariable model adjusting for confounders, a relative decrease in Pa co2 greater than 30% was independently associated with greater odds of neurologic complication (odds ratio [OR], 1.25; 95% CI, 1.07-1.46; p = 0.005). Within this group, with a relative decrease in Pa co2 greater than 30%, the effects of increased relative MAP increased neurologic complications (0.05% per BP Percentile; 95% CI, 0.001-0.11; p = 0.05). CONCLUSIONS: In pediatric patients, a large decrease in Pa co2 and increase in MAP following ECMO initiation are both associated with neurologic complications. Future research focusing on managing these issues carefully soon after ECMO deployment can potentially help to reduce neurologic complications.

Understanding social needs in pregnancy: Prospective validation of a digital short-form screening tool and patient survey.

BACKGROUND: Social determinants of health significantly affect health outcomes, yet are infrequently addressed in prenatal care. OBJECTIVE: This study aimed to improve the efficiency and experience of addressing social needs in pregnancy through: (1) testing a digital short-form screening tool; and (2) characterizing pregnant people's preferences for social needs screening and management. STUDY DESIGN: We developed a digital short-form social determinants of health screening tool from PRAPARE (Protocol for Responding to and Assessing Patients' Assets, Risks, and Experiences), and a survey to query patients' preferences for addressing social needs. Instruments were administered online to peripartum participants, with equal representation of patients with public and private insurance. We calculated the sensitivity and specificity of the short-form tool vs PRAPARE. Quantitative responses were characterized using descriptive statistics. Free-text responses were analyzed with matrix and thematic coding. Survey data were analyzed by subgroups of historically marginalized populations. RESULTS: A total of 215 people completed the survey. Participants were predominantly White (167; 77.7%) and multiparous (145; 67.4%). Unmet social needs were prevalent with both the short-form tool (77.7%) and PRAPARE (96.7%). The sensitivity (79.3%) and specificity (71.4%) of the short-form screener were high for detecting any social need. Most participants believed that it was important for their pregnancy care team to know their social needs (material: 173, 80.5%; support: 200, 93.0%), and over half felt comfortable sharing their needs through in-person or digital modalities if assistance was or was not available (material: 117, 54.4%; support: 122, 56.7%). Free-text themes reflected considerations for integrating social needs in routine prenatal care. Acceptability of addressing social needs in pregnancy was high among all groups. CONCLUSION: A digital short-form social determinants of health screening tool performs well when compared with the gold standard. Pregnant people accept social needs as a part of routine pregnancy care. Future work is needed to operationalize efficient, effective, patient-centered approaches to addressing social needs in pregnancy.

Mapping the Chemical Space of Active-Site Targeted Covalent Ligands for Protein Tyrosine Phosphatases.

Protein tyrosine phosphatases (PTPs) are an important class of enzymes that modulate essential cellular processes through protein dephosphorylation and are dysregulated in various disease states. There is demand for new compounds that target the active sites of these enzymes, for use as chemical tools to dissect their biological roles or as leads for the development of new therapeutics. In this study, we explore an array of electrophiles and fragment scaffolds to investigate the required chemical parameters for covalent inhibition of tyrosine phosphatases. Our analysis juxtaposes the intrinsic electrophilicity of these compounds with their potency against several classical PTPs, revealing chemotypes that inhibit tyrosine phosphatases while minimizing excessive, potentially non-specific reactivity. We also assess sequence divergence at key residues in PTPs to explain their differential susceptibility to covalent inhibition. We anticipate that our study will inspire new strategies to develop covalent probes and inhibitors for tyrosine phosphatases.

High-throughput profiling of sequence recognition by tyrosine kinases and SH2 domains using bacterial peptide display.

Tyrosine kinases and SH2 (phosphotyrosine recognition) domains have binding specificities that depend on the amino acid sequence surrounding the target (phospho)tyrosine residue. Although the preferred recognition motifs of many kinases and SH2 domains are known, we lack a quantitative description of sequence specificity that could guide predictions about signaling pathways or be used to design sequences for biomedical applications. Here, we present a platform that combines genetically encoded peptide libraries and deep sequencing to profile sequence recognition by tyrosine kinases and SH2 domains. We screened several tyrosine kinases against a million-peptide random library and used the resulting profiles to design high-activity sequences. We also screened several kinases against a library containing thousands of human proteome-derived peptides and their naturally-occurring variants. These screens recapitulated independently measured phosphorylation rates and revealed hundreds of phosphosite-proximal mutations that impact phosphosite recognition by tyrosine kinases. We extended this platform to the analysis of SH2 domains and showed that screens could predict relative binding affinities. Finally, we expanded our method to assess the impact of non-canonical and post-translationally modified amino acids on sequence recognition. This specificity profiling platform will shed new light on phosphotyrosine signaling and could readily be adapted to other protein modification/recognition domains.

SEC-TMT facilitates quantitative differential analysis of protein interaction networks.

The majority of cellular proteins interact with at least one partner or assemble into molecular-complexes to exert their function. This network of protein-protein interactions (PPIs) and the composition of macromolecular machines differ between cell types and physiological conditions. Therefore, characterizing PPI networks and their dynamic changes is vital for discovering novel biological functions and underlying mechanisms of cellular processes. However, producing an in-depth, global snapshot of PPIs from a given specimen requires measuring tens to thousands of LC-MS/MS runs. Consequently, while recent works made seminal contributions by mapping PPIs at great depth, almost all focused on just 1-2 conditions, generating comprehensive but mostly static PPI networks. In this study we report the development of SEC-TMT, a method that enables identifying and measuring PPIs in a quantitative manner from only 4-8 LC-MS/MS runs per biological sample. This was accomplished by incorporating tandem mass tag (TMT) multiplexing with a size exclusion chromatography mass spectrometry (SEC-MS) work-flow. SEC-TMT reduces measurement time by an order of magnitude while maintaining resolution and coverage of thousands of cellular interactions, equivalent to the gold standard in the field. We show that SEC-TMT provides benefits for conducting differential analyses to measure changes in the PPI network between conditions. This development makes it feasible to study dynamic systems at scale and holds the potential to drive more rapid discoveries of PPI impact on cellular processes.