Emerging evidence for glucagon-like peptide-1 agonists in slowing chronic kidney disease progression.

PURPOSE OF REVIEW: Diabetic kidney disease continues to increase, and several novel therapeutic agents have been shown to slow the progression of chronic kidney disease in those with diabetes. This review summarizes more recent data on the role of glucagon-like peptide-1 (GLP-1) receptor agonists and kidney outcomes. RECENT FINDINGS: Posthoc analysis of cardiovascular outcome trials, as well as several retrospective studies, demonstrate benefits of GLP-1 receptor agonist therapy for chronic kidney disease progression in diabetics. Although limited randomized clinical trials evidence assessing the effects of GLP-1 receptor agonists on kidney outcomes in diabetic chronic kidney disease patients have been published, FLOW-CKD trial was halted based on interim data for efficacy, and results are awaited. SUMMARY: GLP-1 receptor agonism is a promising therapy for slowing the progression of diabetic chronic kidney disease. Recent studies support kidney benefits GLP-1 receptor agonists over insulin and dipeptidyl peptidase-4-inhibitors, and the FLOW-CKD trial would inform the potential benefits for reducing the need for dialysis and kidney-disease related mortality in those with kidney disease.

SIRPα Mediates IGF1 Receptor in Cardiomyopathy Induced by Chronic Kidney Disease.

BACKGROUND: Chronic kidney disease (CKD) is characterized by increased myocardial mass despite near-normal blood pressure, suggesting the presence of a separate trigger. A potential driver is SIRPα (signal regulatory protein alpha)-a mediator impairing insulin signaling. The objective of this study is to assess the role of circulating SIRPα in CKD-induced adverse cardiac remodeling. METHODS: SIRPα expression was evaluated in mouse models and patients with CKD. Specifically, mutant, muscle-specific, or cardiac muscle-specific SIRPα KO (knockout) mice were examined after subtotal nephrectomy. Cardiac function was assessed by echocardiography. Metabolic responses were confirmed in cultured muscle cells or cardiomyocytes. RESULTS: We demonstrate that SIRPα regulates myocardial insulin/IGF1R (insulin growth factor-1 receptor) signaling in CKD. First, in the serum of both mice and patients, SIRPα was robustly secreted in response to CKD. Second, cardiac muscle upregulation of SIRPα was associated with impaired insulin/IGF1R signaling, myocardial dysfunction, and fibrosis. However, both global and cardiac muscle-specific SIRPα KO mice displayed improved cardiac function when compared with control mice with CKD. Third, both muscle-specific or cardiac muscle-specific SIRPα KO mice did not significantly activate fetal genes and maintained insulin/IGF1R signaling with suppressed fibrosis despite the presence of CKD. Importantly, SIRPα directly interacted with IGF1R. Next, rSIRPα (recombinant SIRPα) protein was introduced into muscle-specific SIRPα KO mice reestablishing the insulin/IGF1R signaling activity. Additionally, overexpression of SIRPα in myoblasts and cardiomyocytes impaired pAKT (phosphorylation of AKT) and insulin/IGF1R signaling. Furthermore, myotubes and cardiomyocytes, but not adipocytes treated with high glucose or cardiomyocytes treated with uremic toxins, stimulated secretion of SIRPα in culture media, suggesting these cells are the origin of circulating SIRPα in CKD. Both intracellular and extracellular SIRPα exert biologically synergistic effects impairing intracellular myocardial insulin/IGF1R signaling. CONCLUSIONS: Myokine SIRPα expression impairs insulin/IGF1R functions in cardiac muscle, affecting cardiometabolic signaling pathways. Circulating SIRPα constitutes an important readout of insulin resistance in CKD-induced cardiomyopathy.

A Multifaceted Hit-Finding Approach Reveals Novel LC3 Family Ligands.

Autophagy-related proteins (Atgs) drive the lysosome-mediated degradation pathway, autophagy, to enable the clearance of dysfunctional cellular components and maintain homeostasis. In humans, this process is driven by the mammalian Atg8 (mAtg8) family of proteins comprising the LC3 and GABARAP subfamilies. The mAtg8 proteins play essential roles in the formation and maturation of autophagosomes and the capture of specific cargo through binding to the conserved LC3-interacting region (LIR) sequence within target proteins. Modulation of interactions of mAtg8 with its target proteins via small-molecule ligands would enable further interrogation of their function. Here we describe unbiased fragment and DNA-encoded library (DEL) screening approaches for discovering LC3 small-molecule ligands. Both strategies resulted in compounds that bind to LC3, with the fragment hits favoring a conserved hydrophobic pocket in mATG8 proteins, as detailed by LC3A-fragment complex crystal structures. Our findings demonstrate that the malleable LIR-binding surface can be readily targeted by fragments; however, rational design of additional interactions to drive increased affinity proved challenging. DEL libraries, which combine small, fragment-like building blocks into larger scaffolds, yielded higher-affinity binders and revealed an unexpected potential for reversible, covalent ligands. Moreover, DEL hits identified possible vectors for synthesizing fluorescent probes or bivalent molecules for engineering autophagic degradation of specific targets.

Soluble Urokinase Receptor and Acute Kidney Injury.

BACKGROUND: Acute kidney injury is common, with a major effect on morbidity and health care utilization. Soluble urokinase plasminogen activator receptor (suPAR) is a signaling glycoprotein thought to be involved in the pathogenesis of kidney disease. We investigated whether a high level of suPAR predisposed patients to acute kidney injury in multiple clinical contexts, and we used experimental models to identify mechanisms by which suPAR acts and to assess it as a therapeutic target. METHODS: We measured plasma levels of suPAR preprocedurally in patients who underwent coronary angiography and patients who underwent cardiac surgery and at the time of admission to the intensive care unit in critically ill patients. We assessed the risk of acute kidney injury at 7 days as the primary outcome and acute kidney injury or death at 90 days as a secondary outcome, according to quartile of suPAR level. In experimental studies, we used a monoclonal antibody to urokinase plasminogen activator receptor (uPAR) as a therapeutic strategy to attenuate acute kidney injury in transgenic mice receiving contrast material. We also assessed cellular bioenergetics and generation of reactive oxygen species in human kidney proximal tubular (HK-2) cells that were exposed to recombinant suPAR. RESULTS: The suPAR level was assessed in 3827 patients who were undergoing coronary angiography, 250 who were undergoing cardiac surgery, and 692 who were critically ill. Acute kidney injury developed in 318 patients (8%) who had undergone coronary angiography. The highest suPAR quartile (vs. the lowest) had an adjusted odds ratio of 2.66 (95% confidence interval [CI], 1.77 to 3.99) for acute kidney injury and 2.29 (95% CI, 1.71 to 3.06) for acute kidney injury or death at 90 days. Findings were similar in the surgical and critically ill cohorts. The suPAR-overexpressing mice that were given contrast material had greater functional and histologic evidence of acute kidney injury than wild-type mice. The suPAR-treated HK-2 cells showed heightened energetic demand and mitochondrial superoxide generation. Pretreatment with a uPAR monoclonal antibody attenuated kidney injury in suPAR-overexpressing mice and normalized bioenergetic changes in HK-2 cells. CONCLUSIONS: High suPAR levels were associated with acute kidney injury in various clinical and experimental contexts. (Funded by the National Institutes of Health and others.).

Interactions between leucines within the signal peptides of megalin and stanniocalcin-1 are crucial for regulation of mitochondrial metabolism.

The mitochondrial intracrine Stanniocalcin 1 (STC1) activates mitochondrial anti-oxidant defenses. LRP2 (megalin) shuttles STC1 to the mitochondria through retrograde early endosome-to-Golgi- and Rab32-mediated pathway, and LRP2 KO impairs mitochondrial respiration and glycolysis. We determined STC1-LRP2 interaction domains using HA- and FLAG-tagged fragments of STC1 and LRP2, respectively, co-expressed in HEK293T cells. The trans-membrane domain of LRP2 is required for trafficking to the mitochondria. STC1-FLAG expressed in LRP2 KO cells fails to reach the mitochondria; thus, mitochondrial STC1 is extracellularly-derived via LRP2-mediated trafficking. Tri-leucines L12-14 in LRP2's signal peptide interact with STC1's signal peptide. Mutant LRP2 (L(12-14)A) does not bind STC1, while hSTC1 lacking signal peptide or Leucines L8/9/11 does not bind LRP2. STC1 fails to induce respiration or glycolysis in megalin KO mouse embryonal fibroblasts (MEF) expressing mutant LRP2, while mutant hSTC1 (L8/L9/L11 - > A8/A9/A11) fails to reach the mitochondria or induce respiration and glycolysis in WT MEF. Our data suggest direct regulation of mitochondrial metabolism by extracellular cues and reveal an important role for signal peptides' leucines in protein-protein interactions and mitochondrial biology.

Exchange of associated factors directs a switch in HBO1 acetyltransferase histone tail specificity.

Histone acetyltransferases (HATs) assemble into multisubunit complexes in order to target distinct lysine residues on nucleosomal histones. Here, we characterize native HAT complexes assembled by the BRPF family of scaffold proteins. Their plant homeodomain (PHD)-Zn knuckle-PHD domain is essential for binding chromatin and is restricted to unmethylated H3K4, a specificity that is reversed by the associated ING subunit. Native BRPF1 complexes can contain either MOZ/MORF or HBO1 as catalytic acetyltransferase subunit. Interestingly, while the previously reported HBO1 complexes containing JADE scaffold proteins target histone H4, the HBO1-BRPF1 complex acetylates only H3 in chromatin. We mapped a small region to the N terminus of scaffold proteins responsible for histone tail selection on chromatin. Thus, alternate choice of subunits associated with HBO1 can switch its specificity between H4 and H3 tails. These results uncover a crucial new role for associated proteins within HAT complexes, previously thought to be intrinsic to the catalytic subunit.

Picomolar zinc binding modulates formation of Bcl10-nucleating assemblies of the caspase recruitment domain (CARD) of CARD9.

The caspase recruitment domain-containing protein 9 (CARD9)-B-cell lymphoma/leukemia 10 (Bcl10) signaling axis is activated in myeloid cells during the innate immune response to a variety of diverse pathogens. This signaling pathway requires a critical caspase recruitment domain (CARD)-CARD interaction between CARD9 and Bcl10 that promotes downstream activation of factors, including NF-κB and the mitogen-activated protein kinase (MAPK) p38. Despite these insights, CARD9 remains structurally uncharacterized, and little mechanistic understanding of its regulation exists. We unexpectedly found here that the CARD in CARD9 binds to Zn2+ with picomolar affinity-a concentration comparable with the levels of readily accessible Zn2+ in the cytosol. NMR solution structures of the CARD9-CARD in the apo and Zn2+-bound states revealed that Zn2+ has little effect on the ground-state structure of the CARD; yet the stability of the domain increased considerably upon Zn2+ binding, with a concomitant reduction in conformational flexibility. Moreover, Zn2+ binding inhibited polymerization of the CARD9-CARD into helical assemblies. Here, we also present a 20-Å resolution negative-stain EM (NS-EM) structure of these filamentous assemblies and show that they adopt a similar helical symmetry as reported previously for filaments of the Bcl10 CARD. Using both bulk assays and direct NS-EM visualization, we further show that the CARD9-CARD assemblies can directly template and thereby nucleate Bcl10 polymerization, a capacity considered critical to propagation of the CARD9-Bcl10 signaling cascade. Our findings indicate that CARD9 is a potential target of Zn2+-mediated signaling that affects Bcl10 polymerization in innate immune responses.

Dynamical network of residue-residue contacts reveals coupled allosteric effects in recognition, catalysis, and mutation.

Detailed understanding of how conformational dynamics orchestrates function in allosteric regulation of recognition and catalysis remains ambiguous. Here, we simulate CypA using multiple-microsecond-long atomistic molecular dynamics in explicit solvent and carry out NMR experiments. We analyze a large amount of time-dependent multidimensional data with a coarse-grained approach and map key dynamical features within individual macrostates by defining dynamics in terms of residue-residue contacts. The effects of substrate binding are observed to be largely sensed at a location over 15 Å from the active site, implying its importance in allostery. Using NMR experiments, we confirm that a dynamic cluster of residues in this distal region is directly coupled to the active site. Furthermore, the dynamical network of interresidue contacts is found to be coupled and temporally dispersed, ranging over 4 to 5 orders of magnitude. Finally, using network centrality measures we demonstrate the changes in the communication network, connectivity, and influence of CypA residues upon substrate binding, mutation, and during catalysis. We identify key residues that potentially act as a bottleneck in the communication flow through the distinct regions in CypA and, therefore, as targets for future mutational studies. Mapping these dynamical features and the coupling of dynamics to function has crucial ramifications in understanding allosteric regulation in enzymes and proteins, in general.

Cyclophilin A catalyzes proline isomerization by an electrostatic handle mechanism.

Proline isomerization is a ubiquitous process that plays a key role in the folding of proteins and in the regulation of their functions. Different families of enzymes, known as "peptidyl-prolyl isomerases" (PPIases), catalyze this reaction, which involves the interconversion between the cis and trans isomers of the N-terminal amide bond of the amino acid proline. However, complete descriptions of the mechanisms by which these enzymes function have remained elusive. We show here that cyclophilin A, one of the most common PPIases, provides a catalytic environment that acts on the substrate through an electrostatic handle mechanism. In this mechanism, the electrostatic field in the catalytic site turns the electric dipole associated with the carbonyl group of the amino acid preceding the proline in the substrate, thus causing the rotation of the peptide bond between the two residues. We identified this mechanism using a combination of NMR measurements, molecular dynamics simulations, and density functional theory calculations to simultaneously determine the cis-bound and trans-bound conformations of cyclophilin A and its substrate as the enzymatic reaction takes place. We anticipate that this approach will be helpful in elucidating whether the electrostatic handle mechanism that we describe here is common to other PPIases and, more generally, in characterizing other enzymatic processes.

Determination of the Full Catalytic Cycle among Multiple Cyclophilin Family Members and Limitations on the Application of CPMG-RD in Reversible Catalytic Systems.

Cyclophilins catalyze cis ↔ trans isomerization of peptidyl-prolyl bonds, influencing protein folding along with a breadth of other biological functions such as signal transduction. Here, we have determined the microscopic rate constants defining the full enzymatic cycle for three human cyclophilins and a more distantly related thermophilic bacterial cyclophilin when catalyzing interconversion of a biologically representative peptide substrate. The cyclophilins studied here exhibit variability in on-enzyme interconversion as well as an up to 2-fold range in rates of substrate binding and release. However, among the human cyclophilins, the microscopic rate constants appear to have been tuned to maintain remarkably similar isomerization rates without a concurrent conservation of apparent binding affinities. While the structures and active site compositions of the human cyclophilins studied here are highly conserved, we find that the enzymes exhibit significant variability in microsecond to millisecond time scale mobility, suggesting a role for the inherent conformational fluctuations that exist within the cyclophilin family as being functionally relevant in regulating substrate interactions. We have additionally modeled the relaxation dispersion profile given by the commonly employed Carr-Purcell-Meiboom-Gill relaxation dispersion (CPMG-RD) experiment when applied to a reversible enzymatic system such as cyclophilin isomerization and identified a significant limitation in the applicability of this approach for monitoring on-enzyme turnover. Specifically, we show both computationally and experimentally that the CPMG-RD experiment is sensitive to noncatalyzed substrate binding and release in reversible systems even at saturating substrate concentrations unless the on-enzyme interconversion rate is much faster than the substrate release rate.

Structure and Dynamics of GeoCyp: A Thermophilic Cyclophilin with a Novel Substrate Binding Mechanism That Functions Efficiently at Low Temperatures.

Thermophilic proteins have found extensive use in research and industrial applications because of their high stability and functionality at elevated temperatures while simultaneously providing valuable insight into our understanding of protein folding, stability, dynamics, and function. Cyclophilins, constituting a ubiquitously expressed family of peptidyl-prolyl isomerases with a range of biological functions and disease associations, have been utilized both for conferring stress tolerances and in exploring the link between conformational dynamics and enzymatic function. To date, however, no active thermophilic cyclophilin has been fully biophysically characterized. Here, we determine the structure of a thermophilic cyclophilin (GeoCyp) from Geobacillus kaustophilus, characterize its dynamic motions over several time scales using an array of methodologies that include chemical shift-based methods and relaxation experiments over a range of temperatures, and measure catalytic activity over a range of temperatures to compare its structure, dynamics, and function to those of a mesophilic counterpart, human cyclophilin A (CypA). Unlike those of most thermophile/mesophile pairs, GeoCyp catalysis is not substantially impaired at low temperatures as compared to that of CypA, retaining ~70% of the activity of its mesophilic counterpart. Examination of substrate-bound ensembles reveals a mechanism by which the two cyclophilins may have adapted to their environments through altering dynamic loop motions and a critical residue that acts as a clamp to regulate substrate binding differentially in CypA and GeoCyp. Fast time scale (pico- to nanosecond) dynamics are largely conserved between the two proteins, in accordance with the high degree of structural similarity, although differences do exist in their temperature dependencies. Slower (microsecond) time scale motions are likewise localized to similar regions in the two proteins with some variability in their magnitudes yet do not exhibit significant temperature dependencies in either enzyme.

Allosteric inhibitors of the Eya2 phosphatase are selective and inhibit Eya2-mediated cell migration.

Eya proteins are essential co-activators of the Six family of transcription factors and contain a unique tyrosine phosphatase domain belonging to the haloacid dehalogenase family of phosphatases. The phosphatase activity of Eya is important for the transcription of a subset of Six1-target genes, and also directs cells to the repair rather than apoptosis pathway upon DNA damage. Furthermore, Eya phosphatase activity has been shown to mediate transformation, invasion, migration, and metastasis of breast cancer cells, making it a potential new drug target for breast cancer. We have previously identified a class of N-arylidenebenzohydrazide compounds that specifically inhibit the Eya2 phosphatase. Herein, we demonstrate that these compounds are reversible inhibitors that selectively inhibit the phosphatase activity of Eya2, but not Eya3. Our mutagenesis results suggest that this class of compounds does not bind to the active site and the binding does not require the coordination with Mg(2+). Moreover, these compounds likely bind within a site on the opposite face of the active site, and function as allosteric inhibitors. We also demonstrate that this class of compounds inhibits Eya2 phosphatase-mediated cell migration, setting the foundation for these molecules to be developed into chemical probes for understanding the specific function of the Eya2 phosphatase and to serve as a prototype for the development of Eya2 phosphatase specific anti-cancer drugs.

Chronic Interstitial Nephritis in Agricultural Communities: Observational and Mechanistic Evidence Supporting the Role of Nephrotoxic Agrochemicals.

Chronic interstitial nephritis in agricultural communities (CINAC) is an epidemic of kidney disease affecting specific tropical and subtropical regions worldwide and is characterized by progressive CKD in the absence of traditional risk factors, such as hypertension and diabetes. CINAC prevalence is higher among young, male agricultural workers, but it also affects women, children, and nonagricultural workers in affected areas. Biopsies from patients with CINAC across regions commonly demonstrate tubular injury with lysosomal aggregates, tubulointerstitial inflammation, and fibrosis and variable glomerular changes. Each endemic area holds environmental risk factors and patient/genetic milieus, resulting in uncertainty about the cause(s) of the disease. Currently, there is no specific treatment available for CINAC. We highlight survey findings of Houston-based migrant workers with CINAC and draw similarities between kidney injury phenotype of patients with CINAC and mice treated chronically with paraquat, an herbicide used worldwide. We propose potential pathways and mechanisms for kidney injury in patients with CINAC, which may offer clues for potential therapies.

Discovery and Clinical Proof-of-Concept of RLY-2608, a First-in-Class Mutant-Selective Allosteric PI3Kα Inhibitor That Decouples Antitumor Activity from Hyperinsulinemia.

PIK3CA (PI3Kα) is a lipid kinase commonly mutated in cancer, including ∼40% of hormone receptor-positive breast cancer. The most frequently observed mutants occur in the kinase and helical domains. Orthosteric PI3Kα inhibitors suffer from poor selectivity leading to undesirable side effects, most prominently hyperglycemia due to inhibition of wild-type (WT) PI3Kα. Here, we used molecular dynamics simulations and cryo-electron microscopy to identify an allosteric network that provides an explanation for how mutations favor PI3Kα activation. A DNA-encoded library screen leveraging electron microscopy-optimized constructs, differential enrichment, and an orthosteric-blocking compound led to the identification of RLY-2608, a first-in-class allosteric mutant-selective inhibitor of PI3Kα. RLY-2608 inhibited tumor growth in PIK3CA-mutant xenograft models with minimal impact on insulin, a marker of dysregulated glucose homeostasis. RLY-2608 elicited objective tumor responses in two patients diagnosed with advanced hormone receptor-positive breast cancer with kinase or helical domain PIK3CA mutations, with no observed WT PI3Kα-related toxicities. SIGNIFICANCE: Treatments for PIK3CA-mutant cancers are limited by toxicities associated with the inhibition of WT PI3Kα. Molecular dynamics, cryo-electron microscopy, and DNA-encoded libraries were used to develop RLY-2608, a first-in-class inhibitor that demonstrates mutant selectivity in patients. This marks the advance of clinical mutant-selective inhibition that overcomes limitations of orthosteric PI3Kα inhibitors. See related commentary by Gong and Vanhaesebroeck, p. 204 . See related article by Varkaris et al., p. 227 . This article is featured in Selected Articles from This Issue, p. 201.

Evaluation of an Academic Detailing Program to Improve Blood Pressure Measurement and Hypertension Treatment in Urban Community Health Centers.

INTRODUCTION: Hypertension is estimated at a prevalence of 30 to 50% in the United States. Only 54% of patients with hypertension have their condition adequately controlled. This study aimed to use academic detailing (AD) to improve practice team knowledge and confidence in blood pressure (BP) management, specifically in low-income, underresourced patient populations. METHODS: AD was developed for five practices that care for high percentages of Medicaid-eligible patients. A needs assessment was administered to each site's practice champion to determine high-yield AD sessions for their team. Presession and postsession evaluations were completed by practice team members. RESULTS: Fifty providers and eight staff member participants completed evaluations over nine sessions for two AD topics at four practice sites. Statistical significance was found for several items within sessions including accurately choosing cuff sizes and identifying barriers to home BP monitoring. DISCUSSION: AD is a potentially financially efficient model to improve knowledge and confidence in hypertension care at the practice level. Tailoring session content to specific needs of a practice site, along with an identified practice champion, facilitated implementation of the program. This replicable model is one way to deliver evidence-based information to those who serve Medicaid-eligible patients.

Treatment of Severe COVID-19 Infection With Remdesivir in Peritoneal Dialysis.

End-stage kidney disease (ESKD) has been shown to be correlated with an increased risk of COVID-19 infection and mortality. Remdesivir is an effective non-EUA U.S. Food and Drug Administration (FDA)-approved antiviral agent for the treatment of COVID-19 in hospitalized adult and pediatric patients, though a lack of data has prevented its use in patients with severe kidney disease including dialysis patients. Some observational studies report the use of remdesivir in hemodialysis patients, but there are no reports of patients treated with remdesivir on peritoneal dialysis. Dialysis modalities may affect drug pharmacokinetics, and safety and efficiency of remdesivir in peritoneal dialysis is unknown. We report the first case, to our knowledge, of using remdesivir in a patient treated with peritoneal dialysis with no significant adverse events. This case illustrates the potential for remdesivir to be considered in peritoneal dialysis patients with severe COVID infection. Proper risk analysis and careful monitoring should be done, given the unpredictable clearance of the drug.

Postoperative Gabapentin's Effect on Opioid Consumption and Pain Control Following Sinonasal Surgery.

OBJECTIVE: This study investigates the impact of postoperative gabapentin on opioid consumption and pain control following endoscopic sinus surgery (ESS) and/or septoplasty. METHODS: Patients who underwent ESS and/or septoplasty at a single institution from 2021 to 2022 were enrolled. All patients received postoperative hydrocodone-acetaminophen for pain control. Half of the patients were also prescribed gabapentin for the first postoperative day in addition to hydrocodone-acetaminophen. Subjects completed the Revised American Pain Society Patient Outcome Questionnaire 24 h and 7 days after surgery. We conducted a multivariable regression analysis to assess opioid consumption and improvement in pain scores in the first week between gabapentin and non-gabapentin groups. RESULTS: A total of 102 subjects, 51 in each arm, were enrolled. The mean age was 52 years and 53% of participants were female. Controlling for important baseline demographic, clinical, and surgically related variables, the addition of postoperative gabapentin was associated with a 44% (9.5 mg from 21.6 mg) reduction in opioids consumed in the first postoperative week (B = -9.54, 95% C.I. = [-17.84, -1.24], p = 0.025). In addition, patients in both arms exhibited similar improvement in pain severity and sleep interference in the first 7 days (B = -1.59, 95% C.I. = [-5.03, 1.84], p = 0.36). CONCLUSION: To the best of our knowledge, this is the first study to investigate the impact of postoperative gabapentin on opioid consumption and pain control following ESS and/or septoplasty. Our analysis demonstrated that postoperative gabapentin effectively reduced opioid use during the first postoperative week without compromising pain control. LEVEL OF EVIDENCE: 3 Laryngoscope, 133:1065-1072, 2023.

Simulation-based workshop for emergency preparedness in otolaryngology.

Objectives: This study aimed to evaluate the outcomes of a hands-on simulation-based course with emphasis on procedural techniques, clinical reasoning, and communication skills developed to improve junior Otolaryngology - Head and Neck Surgery (OHNS) residents' preparedness in managing otolaryngologic emergencies. Methods: Junior OHNS residents and faculty from residency programs in California, Nevada, and Arizona participated in this workshop in 2020 and 2021. The stations featured airway management techniques, ultrasound-guided needle aspiration, nasoseptal hematoma evacuation, and facial fracture repair using various models and cadavers. Participants completed a pre-workshop survey, post-workshop survey, and 2-month follow-up survey that assessed resident anxiety and confidence in three OHNS emergency situations across knowledge, manual skills, and teamwork using a 5-point Likert scale. Results: Pre-workshop surveys reported the least anxiety and most confidence in teamwork, but the most anxiety and least confidence in technical skills and knowledge related to foreign body retrieval and airway management. Immediately post-workshop participants reported significant reductions in anxiety and increases in confidence, largest in the manual skills domain, in foreign body retrieval (anxiety: -0.99, confidence: +0.95, p < .01) and airway management stations (anxiety: -0.68, confidence: +1.07, p < .01). Data collected for the epistaxis station showed decreasing confidence and increasing anxiety following the workshop. Conclusion: Our findings demonstrate the effectiveness of a workshop in preparing junior residents in potentially lifesaving otolaryngologic techniques that residents will encounter. Optimizing use of simulation centered training can inform the future of residency education, improving confidence and decreasing anxiety in residents responsible for the safety of patients. Level of Evidence: III.

Extracellular cyclophilin-A stimulates ERK1/2 phosphorylation in a cell-dependent manner but broadly stimulates nuclear factor kappa B.

BACKGROUND: Although the peptidyl-prolyl isomerase, cyclophilin-A (peptidyl-prolyl isomerase, PPIA), has been studied for decades in the context of its intracellular functions, its extracellular roles as a major contributor to both inflammation and multiple cancers have more recently emerged. A wide range of activities have been ascribed to extracellular PPIA that include induction of cytokine and matrix metalloproteinase (MMP) secretion, which potentially underlie its roles in inflammation and tumorigenesis. However, there have been conflicting reports as to which particular signaling events are under extracellular PPIA regulation, which may be due to either cell-dependent responses and/or the use of commercial preparations recently shown to be highly impure. METHODS: We have produced and validated the purity of recombinant PPIA in order to subject it to a comparative analysis between different cell types. Specifically, we have used a combination of multiple methods such as luciferase reporter screens, translocation assays, phosphorylation assays, and nuclear magnetic resonance to compare extracellular PPIA activities in several different cell lines that included epithelial and monocytic cells. RESULTS: Our findings have revealed that extracellular PPIA activity is cell type-dependent and that PPIA signals via multiple cellular receptors beyond the single transmembrane receptor previously identified, Extracellular Matrix MetalloPRoteinase Inducer (EMMPRIN). Finally, while our studies provide important insight into the cell-specific responses, they also indicate that there are consistent responses such as nuclear factor kappa B (NFκB) signaling induced in all cell lines tested. CONCLUSIONS: We conclude that although extracellular PPIA activates several common pathways, it also targets different receptors in different cell types, resulting in a complex, integrated signaling network that is cell type-specific.