ZAP-70 in Signaling, Biology, and Disease.

T cells possess an array of functional capabilities important for host defense against pathogens and tumors. T cell effector functions require the T cell antigen receptor (TCR). The TCR has no intrinsic enzymatic activity, and thus signal transduction from the receptor relies on additional signaling molecules. One such molecule is the cytoplasmic tyrosine kinase ZAP-70, which associates with the TCR complex and is required for initiating the canonical biochemical signal pathways downstream of the TCR. In this article, we describe recent structure-based insights into the regulation and substrate specificity of ZAP-70, and then we review novel methods for determining the role of ZAP-70 catalytic activity-dependent and -independent signals in developing and mature T cells. Lastly, we discuss the disease states in mouse models and humans, which range from immunodeficiency to autoimmunity, that are caused by mutations in ZAP-70.

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.

Slow phosphorylation of a tyrosine residue in LAT optimizes T cell ligand discrimination.

Self-non-self discrimination is central to T cell-mediated immunity. The kinetic proofreading model can explain T cell antigen receptor (TCR) ligand discrimination; however, the rate-limiting steps have not been identified. Here, we show that tyrosine phosphorylation of the T cell adapter protein LAT at position Y132 is a critical kinetic bottleneck for ligand discrimination. LAT phosphorylation at Y132, mediated by the kinase ZAP-70, leads to the recruitment and activation of phospholipase C-γ1 (PLC-γ1), an important effector molecule for T cell activation. The slow phosphorylation of Y132, relative to other phosphosites on LAT, is governed by a preceding glycine residue (G131) but can be accelerated by substituting this glycine with aspartate or glutamate. Acceleration of Y132 phosphorylation increases the speed and magnitude of PLC-γ1 activation and enhances T cell sensitivity to weaker stimuli, including weak agonists and self-peptides. These observations suggest that the slow phosphorylation of Y132 acts as a proofreading step to facilitate T cell ligand discrimination.

Lck promotes Zap70-dependent LAT phosphorylation by bridging Zap70 to LAT.

T cell-antigen receptor (TCR) signaling requires the sequential activities of the kinases Lck and Zap70. Upon TCR stimulation, Lck phosphorylates the TCR, thus leading to the recruitment, phosphorylation, and activation of Zap70. Lck binds and stabilizes phosho-Zap70 by using its SH2 domain, and Zap70 phosphorylates the critical adaptors LAT and SLP76, which coordinate downstream signaling. It is unclear whether phosphorylation of these adaptors occurs through passive diffusion or active recruitment. We report the discovery of a conserved proline-rich motif in LAT that mediates efficient LAT phosphorylation. Lck associates with this motif via its SH3 domain, and with phospho-Zap70 via its SH2 domain, thereby acting as a molecular bridge that facilitates the colocalization of Zap70 and LAT. Elimination of this proline-rich motif compromises TCR signaling and T cell development. These results demonstrate the remarkable multifunctionality of Lck, wherein each of its domains has evolved to orchestrate a distinct step in TCR signaling.

Glucocorticoid receptor confers resistance to antiandrogens by bypassing androgen receptor blockade.

The treatment of advanced prostate cancer has been transformed by novel antiandrogen therapies such as enzalutamide. Here, we identify induction of glucocorticoid receptor (GR) expression as a common feature of drug-resistant tumors in a credentialed preclinical model, a finding also confirmed in patient samples. GR substituted for the androgen receptor (AR) to activate a similar but distinguishable set of target genes and was necessary for maintenance of the resistant phenotype. The GR agonist dexamethasone was sufficient to confer enzalutamide resistance, whereas a GR antagonist restored sensitivity. Acute AR inhibition resulted in GR upregulation in a subset of prostate cancer cells due to relief of AR-mediated feedback repression of GR expression. These findings establish a mechanism of escape from AR blockade through expansion of cells primed to drive AR target genes via an alternative nuclear receptor upon drug exposure.

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

Mutations in the tyrosine phosphatase Src homology-2 domain-containing protein tyrosine phosphatase-2 (SHP2) are associated with a variety of human diseases. Most mutations in SHP2 increase its basal catalytic activity by disrupting autoinhibitory 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 to 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.

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.

Inhibition of EZH2 transactivation function sensitizes solid tumors to genotoxic stress.

Drugs that block the activity of the methyltransferase EZH2 are in clinical development for the treatment of non-Hodgkin lymphomas harboring EZH2 gain-of-function mutations that enhance its polycomb repressive function. We have previously reported that EZH2 can act as a transcriptional activator in castration-resistant prostate cancer (CRPC). Now we show that EZH2 inhibitors can also block the transactivation activity of EZH2 and inhibit the growth of CRPC cells. Gene expression and epigenomics profiling of cells treated with EZH2 inhibitors demonstrated that in addition to derepressing gene expression, these compounds also robustly down-regulate a set of DNA damage repair (DDR) genes, especially those involved in the base excision repair (BER) pathway. Methylation of the pioneer factor FOXA1 by EZH2 contributes to the activation of these genes, and interaction with the transcriptional coactivator P300 via the transactivation domain on EZH2 directly turns on the transcription. In addition, CRISPR-Cas9-mediated knockout screens in the presence of EZH2 inhibitors identified these BER genes as the determinants that underlie the growth-inhibitory effect of EZH2 inhibitors. Interrogation of public data from diverse types of solid tumors expressing wild-type EZH2 demonstrated that expression of DDR genes is significantly correlated with EZH2 dependency and cellular sensitivity to EZH2 inhibitors. Consistent with these findings, treatment of CRPC cells with EZH2 inhibitors dramatically enhances their sensitivity to genotoxic stress. These studies reveal a previously unappreciated mechanism of action of EZH2 inhibitors and provide a mechanistic basis for potential combination cancer therapies.

Crafting Unnatural Peptide Macrocycles via Rh(III)-Catalyzed Carboamidation.

Contemporary developments in the field of peptide macrocyclization methodology are imperative for enabling the advance of drug design in medicinal chemistry. This report discloses a Rh(III)-catalyzed macrocyclization via carboamidation, reacting acryloyl-peptide-dioxazolone precursors and arylboronic acids to form complex cyclic peptides with concomitant incorporation of noncanonical α-amino acids. The diverse and modular technology allows for expedient access to a wide variety of cyclic peptides from 4 to 15 amino acids in size and features simultaneous formation of unnatural phenylalanine and tyrosine derivatives with up to >20:1 diastereoselectivity. The reaction showcases an expansive substrate scope with 45 examples and is compatible with the majority of standard protected amino acids used in Fmoc-solid phase peptide synthesis. The methodology is applied to the synthesis of multiple peptidomimetic macrocyclic analogs, including derivatives of cyclosomatostatin and gramicidin S.

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.

Pediatric Hematology and Oncology Patients on Extracorporeal Membrane Oxygenation: Outcomes in a Multicenter, Retrospective Cohort 2009-2021.

OBJECTIVE: To describe characteristics associated with survival for pediatric patients with an oncologic diagnosis or hematopoietic cell transplant (HCT) supported with extracorporeal membrane oxygenation (ECMO). DESIGN: Multicenter, retrospective study. SETTING: Sixteen PICUs in the United States and Israel. PATIENTS: We included patients aged younger than 19 years with an oncologic diagnosis or HCT who required ECMO support between 2009 and 2021. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: A total of 149 patients were included in the study cohort. There were 118 patients with an oncologic diagnosis and 31 that received HCT. The indications for ECMO were respiratory failure (46%), combined respiratory and cardiac failure (28%), and cardiac failure (25%). Venovenous (V-V) ECMO was used in 45% of patients, with 53% of patients being placed on venoarterial (V-A) ECMO. For oncologic and HCT groups, survival to ECMO decannulation was 52% (62/118) and 64% (20/31), and survival to hospital discharge was 36% (43/118) and 42% (13/31), respectively. After adjusting for other factors, requiring cardiopulmonary resuscitation was associated with greater odds ratio of mortality (3.0 [95% CI, 1.2-7.7]). CONCLUSIONS: Survival to ECMO decannulation of pediatric oncologic and HCT patients in this study was 52-64%, depending upon diagnosis. However, survival to hospital discharge remains poor. Future research should prioritize understanding factors contributing to this survival gap within these patient populations.

Treatment of Symptomatic Carotid Webs: A Single-Center Review.

BACKGROUND: 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 to 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. CONCLUSIONS: 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.

Modular Synthesis of Unnatural Peptides via Rh(III)-Catalyzed Diastereoselective Three-Component Carboamidation Reaction.

Herein we report a modular peptide ligation methodology that couples dioxazolones, arylboronic acids, and acrylamides to construct amide bonds in a diastereoselective manner under mild conditions, facilitated by Rh(III) catalysis. By converting the C-terminus of one peptide into a dioxazolone and the N-terminus of a second peptide into an acrylamide, the two pieces can be bridged by an arylboronic acid to construct unnatural phenylalanine, tyrosine, and tryptophan residues at the junction point with diastereoselectivity for their corresponding d-stereocenters. The reaction exhibits excellent functional group tolerance with a large substrate scope and is compatible with a wide array of protected amino acid residues that are utilized in Fmoc solid phase peptide synthesis. The methodology is applied to the synthesis of six diastereomeric proteasome inhibitor analogs, as well as the ligation of two 10-mer oligopeptides to construct a 21-mer polypeptide with an unnatural phenylalanine residue at the center.

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.

The use of machine learning and artificial intelligence within pediatric critical care.

The field of pediatric critical care has been hampered in the era of precision medicine by our inability to accurately define and subclassify disease phenotypes. This has been caused by heterogeneity across age groups that further challenges the ability to perform randomized controlled trials in pediatrics. One approach to overcome these inherent challenges include the use of machine learning algorithms that can assist in generating more meaningful interpretations from clinical data. This review summarizes machine learning and artificial intelligence techniques that are currently in use for clinical data modeling with relevance to pediatric critical care. Focus has been placed on the differences between techniques and the role of each in the clinical arena. The various forms of clinical decision support that utilize machine learning are also described. We review the applications and limitations of machine learning techniques to empower clinicians to make informed decisions at the bedside. IMPACT: Critical care units generate large amounts of under-utilized data that can be processed through artificial intelligence. This review summarizes the machine learning and artificial intelligence techniques currently being used to process clinical data. The review highlights the applications and limitations of these techniques within a clinical context to aid providers in making more informed decisions at the bedside.

ERF mutations reveal a balance of ETS factors controlling prostate oncogenesis.

Half of all prostate cancers are caused by the TMPRSS2-ERG gene-fusion, which enables androgens to drive expression of the normally silent E26 transformation-specific (ETS) transcription factor ERG in prostate cells. Recent genomic landscape studies of such cancers have reported recurrent point mutations and focal deletions of another ETS member, the ETS2 repressor factor ERF. Here we show these ERF mutations cause decreased protein stability and mostly occur in tumours without ERG upregulation. ERF loss recapitulates the morphological and phenotypic features of ERG gain in normal mouse prostate cells, including expansion of the androgen receptor transcriptional repertoire, and ERF has tumour suppressor activity in the same genetic background of Pten loss that yields oncogenic activity by ERG. In the more common scenario of ERG upregulation, chromatin immunoprecipitation followed by sequencing indicates that ERG inhibits the ability of ERF to bind DNA at consensus ETS sites both in normal and in cancerous prostate cells. Consistent with a competition model, ERF overexpression blocks ERG-dependent tumour growth, and ERF loss rescues TMPRSS2-ERG-positive prostate cancer cells from ERG dependency. Collectively, these data provide evidence that the oncogenicity of ERG is mediated, in part, by competition with ERF and they raise the larger question of whether other gain-of-function oncogenic transcription factors might also inactivate endogenous tumour suppressors.

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.

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.