Exploring Product Release from Yeast Cytosine Deaminase with Metadynamics.

The yeast cytosine deaminase (yCD) enzyme/5-fluorocytosine prodrug system is a promising candidate for targeted chemotherapeutics. After conversion of the prodrug into the toxic chemotherapeutic drug, 5-fluorouracil (5-FU), the slow product release from the enzyme limits the overall catalytic efficiency of the enzyme/prodrug system. Here, we present a computational study of the product release of the anticancer drug, 5-FU, from yCD using metadynamics. We present a comparison of the 5-FU drug to the natural enzyme product, uracil. We use volume-based metadynamics to compute the free energy landscape for product release and show a modest binding affinity for the product to the enzyme, consistent with experiments. Next, we use infrequent metadynamics to estimate the unbiased release rate from Kramers time-dependent rate theory and find a favorable comparison to experiment with a slower rate of product release for the 5-FU system. Our work demonstrates how adaptive sampling methods can be used to study the protein-ligand unbinding process for engineering enzyme/prodrug systems and gives insights into the molecular mechanism of product release for the yCD/5-FU system.

Chikungunya virus virus-like particle vaccine is well tolerated and immunogenic in chikungunya seropositive individuals.

In a phase 2 safety and immunogenicity study of a chikungunya virus virus-like particle (CHIKV VLP) vaccine in an endemic region, of 400 total participants, 78 were found to be focus reduction neutralizing antibody seropositive at vaccination despite being ELISA seronegative at screening, of which 39 received vaccine. This post hoc analysis compared safety and immunogenicity of CHIKV VLP vaccine in seropositive (n = 39) versus seronegative (n = 155) vaccine recipients for 72 weeks post-vaccination. There were no differences in solicited adverse events, except injection site swelling in 10.3% of seropositive versus 0.6% of seronegative recipients (p = 0.006). Baseline seropositive vaccine recipients had stronger post-vaccination luciferase neutralizing antibody responses versus seronegative recipients (peak geometric mean titer of 3594 and 1728, respectively) persisting for 72 weeks, with geometric mean fold increases of 3.1 and 13.2, respectively. In this small study, CHIKV VLP vaccine was well-tolerated and immunogenic in individuals with pre-existing immunity. ClinicalTrials.gov Identifier: NCT02562482.

Pediatric Laryngeal Coccidioidomycosis: A Case Series in an Endemic Region.

BACKGROUND: Laryngeal coccidioidomycosis is a rare but life-threatening manifestation of coccidioidomycosis. Data in children are sparse and limited to case reports. We conducted this study to review the characteristics of laryngeal coccidioidomycosis in children. METHODS: We performed a retrospective review of patients ≤21 years of age with laryngeal coccidioidomycosis who were treated from January 2010 to December 2017. We collected demographic data, clinical and laboratory studies and patient outcomes. RESULTS: Five cases of pediatric laryngeal coccidioidomycosis were reviewed. All children were Hispanic and 3 were female. The median age was 1.8 years and the median duration of symptoms before diagnosis was 24 days. The most common symptoms included fever (100%), stridor (60%), cough (100%) and vocal changes (40%). Airway obstruction requiring tracheostomy and/or intubation for airway management was present in 80%. The most frequent location of lesions was the subglottic area. Coccidioidomycosis complement fixation titers were frequently low and culture/histopathology of laryngeal tissue was necessary to make a definitive diagnosis. All patients required surgical debridement and were treated with antifungal agents. None of the patients had recurrence during the follow-up period. CONCLUSIONS: This study suggests that laryngeal coccidioidomycosis in children presents with refractory stridor or dysphonia and severe airway obstruction. Favorable outcomes can be achieved with a comprehensive diagnostic work-up and aggressive surgical and medical management. With the rise in cases of coccidioidomycosis, physicians should have a heightened awareness regarding the possibility of laryngeal coccidioidomycosis when encountering children who have visited or reside in endemic areas with stridor or dysphonia.

Immunogenicity of partial doses of live oral cholera vaccine CVD 103-HgR in children in the United States.

In a phase 4, placebo-controlled, double-blind, multi-center study performed to assess the immunogenicity of a single oral dose of live, attenuated cholera vaccine, volunteers aged 2-17 years were randomized 6:1 to receive 1 × 109 colony forming units of PXVX0200 or placebo. In the subset of subjects who consumed < 80 % of the vaccine dose, seroconversion rates were calculated and stratified by amount consumed. Of 468 subjects dosed, a subset of 33 (7 %) received < 80 % of the vaccine dose. SVA seroconversion occurred in 75.8 % of these subjects, including 100 % (7/7) of those who took 50-80 % and 69.2 % (18/26) of those who took < 50 %, versus 98.5 % of those who consumed 80 % or more. Vaccination with PXVX0200 produced an immune response in most children who received partial dosing. Since SVA seroconversion is a strong correlate of protection, PXVX0200 may protect against cholera infection in children who ingest only part of the vaccine dose.

Central Nervous System Coccidioidomycosis in Children: A Retrospective Case Series.

BACKGROUND: Published literature on central nervous system (CNS) coccidioidomycosis in children is limited. Here we describe a large case series of pediatric CNS coccidioidomycosis from a tertiary care center in an endemic region. METHOD: This is a retrospective case review of patients ≤21 years old with a diagnosis of CNS coccidioidomycosis from January 1, 2000, to December 31, 2018. RESULTS: Thirty patients (median age 10.8 years) were identified and most (93%) were previously healthy. Fever (90%), headache (70%), vomiting (53%), and fatigue (57%) were the most common presenting clinical manifestations, with focal neurological signs/symptoms present in 14 (47%). The initial serum Coccidioides compliment fixation (CF) titer was ≤ 1:8 in 33%. Most patients had extra-axial brain involvement (83%) and seven (23%) had associated spinal cord disease. Shunt placement was required in 70% and 62% required revision. Fluconazole was the initial treatment in 22 (73%), with treatment failure occurring in 50%. Most patients (77%) stabilized and were maintained on suppressive therapy, 4 (13%) experienced relapses and/or progressive disease, and one (3%) died, while long-term neurological complications occurred in 17%. CONCLUSIONS: CNS coccidioidomycosis is an uncommon and sometimes devastating complication of disseminated coccidioidomycosis. Many patients present with relatively low CF titers and hydrocephalus is common. Fluconazole treatment failures are common, and management remains difficult despite recent advances in therapy. Most patients do well once the disease is stabilized and require lifelong therapy. Newer therapeutic agents are needed.

Field-Theoretic Simulation Method to Study the Liquid-Liquid Phase Separation of Polymers.

Liquid-liquid phase separation (LLPS) is a process that results in the formation of a polymer-rich liquid phase coexisting with a polymer-depleted liquid phase. LLPS plays a critical role in the cell through the formation of membrane-less organelles, but it also has a number of biotechnical and biomedical applications such as drug confinement and its targeted delivery. In this chapter, we present a computational efficient methodology that uses field-theoretic simulations (FTS) with complex Langevin (CL) sampling to characterize polymer phase behavior and delineate the LLPS phase boundaries. This approach is a powerful complement to analytical and explicit-particle simulations, and it can serve to inform experimental LLPS studies. The strength of the method lies in its ability to properly sample a large ensemble of polymers in a saturated solution while including the effect of composition fluctuations on LLPS. We describe the approaches that can be used to accurately construct phase diagrams of a variety of molecularly designed polymers and illustrate the method by generating an approximation-free phase diagram for a classical symmetric diblock polyampholyte.

Structures of Streptococcus pyogenes class A sortase in complex with substrate and product mimics provide key details of target recognition.

The cell wall is a critical extracellular barrier for bacteria and many other organisms. In bacteria, this structural layer consists of peptidoglycan, which maintains cell shape and structural integrity and provides a scaffold for displaying various protein factors. To attach proteins to the cell wall, Gram-positive bacteria utilize sortase enzymes, which are cysteine transpeptidases that recognize and cleave a specific sorting signal, followed by ligation of the sorting signal-containing protein to the peptidoglycan precursor lipid II (LII). This mechanism is the subject of considerable interest as a target for therapeutic intervention and as a tool for protein engineering, where sortases have enabled sortase-mediated ligation or sortagging strategies. Despite these uses, there remains an incomplete understanding of the stereochemistry of substrate recognition and ligation product formation. Here, we solved the first structures of sortase A from Streptococcus pyogenes bound to two substrate sequences, LPATA and LPATS. In addition, we synthesized a mimetic of the product of sortase-mediated ligation involving LII (LPAT-LII) and solved the complex structure in two ligand conformations. These structures were further used as the basis for molecular dynamics simulations to probe sortase A-ligand dynamics and to construct a model of the acyl-enzyme intermediate, thus providing a structural view of multiple key states in the catalytic mechanism. Overall, this structural information provides new insights into the recognition of the sortase substrate motif and LII ligation partner and will support the continued development of sortases for protein engineering applications.

Safety and immunogenicity of PXVX0317, an aluminium hydroxide-adjuvanted chikungunya virus-like particle vaccine: a randomised, double-blind, parallel-group, phase 2 trial.

BACKGROUND: Chikungunya virus (CHIKV) disease is an ongoing public health threat. We aimed to evaluate the safety and immunogenicity of PXVX0317, an aluminium hydroxide-adjuvanted formulation of a CHIKV virus-like particle (VLP) vaccine. METHODS: This randomised, double-blind, parallel-group, phase 2 trial was conducted at three clinical trial centres in the USA. Eligible participants were healthy CHIKV-naïve adults aged 18-45 years. Participants were stratified by site and randomly assigned (1:1:1:1:1:1:1:1) to one of the eight vaccination groups using a block size of 16. Group 1 received two doses of unadjuvanted PXVX0317 28 days apart (2 × 20 µg; standard); all other groups received adjuvanted PXVX0317: groups 2-4 received two doses 28 days apart (2 × 6 µg [group 2], 2 × 10 µg [group 3], or 2 × 20 µg [group 4]; standard); group 4 also received a booster dose 18 months after the first active injection (40 µg; standard plus booster); groups 5-7 received two doses 14 days apart (2 × 6 µg [group 5], 2 × 10 µg [group 6], or 2 × 20 µg [group 7]; accelerated); and group 8 received one dose (1 × 40 µg; single). The primary endpoint was the geometric mean titre of anti-CHIKV neutralising antibody on day 57 (28 days after the last vaccination), assessed in the immunogenicity-evaluable population. Additionally, we assessed safety. This trial is registered at ClinicalTrials.gov, NCT03483961. FINDINGS: This trial was conducted from April 18, 2018, to Sept 21, 2020; 468 participants were assessed for eligibility. Of these, 415 participants were randomly assigned to eight groups (n=53 in groups 1, 5, and 6; n=52 in groups 2 and 8; n=51 in groups 3 and 7; and n=50 in group 4) and 373 were evaluable for immunogenicity. On day 57, serum neutralising antibody geometric mean titres were 2057·0 (95% CI 1584·8-2670·0) in group 1, 1116·2 (852·5-1461·4; p=0·0015 vs group 1 used as a reference) in group 2, 1465·3 (1119·1-1918·4; p=0·076) in group 3, 2023·8 (1550·5-2641·7; p=0·93) in group 4, 920·1 (710·9-1190·9; p<0·0001) in group 5, 1206·9 (932·4-1562·2; p=0·0045) in group 6, 1562·8 (1204·1-2028·3; p=0·14) in group 7, and 1712·5 (1330·0-2205·0; p=0·32) in group 8. In group 4, a booster dose increased serum neutralising antibody geometric mean titres from 215·7 (95% CI 160·9-289·1) on day 547 to 10 941·1 (7378·0-16 225·1) on day 575. Durability of the immune response (evaluated in groups 1, 4, and 8) was shown up to 2 years. The most common solicited adverse event was pain at the injection site, reported in 12 (23%) of 53 participants who received the unadjuvanted vaccine (group 1) and 111 (31%) of 356 who received the adjuvanted vaccine. No vaccine-related serious adverse events were reported. INTERPRETATION: PXVX0317 was well tolerated and induced a robust and durable serum neutralising antibody immune response against CHIKV up to 2 years. A single 40 µg injection of adjuvanted PXVX0317 is being further investigated in phase 3 clinical trials (NCT05072080 and NCT05349617). FUNDING: Emergent BioSolutions.

Pediatric Musculoskeletal Coccidioidomycosis in Central California: A Single Center Experience.

BACKGROUND: Published literature on musculoskeletal coccidioidomycosis is sparse and limited to case reports and case series. This is one of the largest case series to describe the clinical presentation, diagnosis, medical and surgical management and outcomes of pediatric musculoskeletal coccidioidomycosis at a tertiary care hospital. METHOD: A retrospective case review was performed on patients ≤ 21 years old who were followed at a tertiary care center with a diagnosis of musculoskeletal coccidioidomycosis from January 1, 2007, to December 31, 2020. Descriptive data are expressed as medians and interquartile range (IQR) for continuous variables or as frequency and percentage for categorical variables. Categorical values were compared using the χ2 test. RESULTS: Forty-one patients were identified. The median age was 12.8 years, and most were male (71%), Latinx (66%) and healthy (71%). Limb swelling (66%), bone pain (54%) and joint pain (46%) were the most common presenting symptoms. Multiple bone involvement was present in 29% while 12% had the joint disease, and craniofacial (n = 10) and metacarpal/metatarsal bones (n=9) were the most commonly involved sites. Elevated Coccidioides complement fixation (CF) titers ≥1:32 were seen in 90% of the patients. Thirty-three patients (81%) required surgical interventions and of these 16 (48%) required additional surgical procedures. Eleven patients (27%) had disease relapse. Children >13 years of age were more likely to have > 1 organ involvement (16 vs. 7, P = 0.04), multiple bone involvement (10 vs. 2, P = 0.004) and maximum Coccidioides CF titers >1:128 (13 vs. 6, P = 0.02). CONCLUSIONS: In endemic areas, musculoskeletal coccidioidomycosis causes a substantial disease burden in children and should be considered in the differential diagnosis of those presenting with bone and joint pain or swelling. Early diagnosis and treatment are essential to minimize long-term morbidity and mortality.

Neonatal Coccidioidomycosis: A Single-center Experience and Review of the Literature.

BACKGROUND: Coccidioidomycosis is common in adult and pediatric populations living in endemic areas of the United States but has rarely been reported in neonates. We reviewed recent cases of neonatal coccidioidomycosis treated at a tertiary care children's hospital in an endemic area and compared them with previously reported cases in the literature. METHODS: We performed a retrospective chart review of infants 1 month old or less hospitalized with a diagnosis of coccidioidomycosis from January 1, 2014, to December 31, 2019. Additionally, we performed a literature review of all reported cases of neonatal coccidioidomycosis over the past 7 decades through PubMed. Infants born to mothers with confirmed or suspected active coccidioidomycosis were excluded. RESULTS: Three cases of neonatal coccidioidomycosis were identified at our institution. Each presented in a unique manner and had an alternative diagnosis at the time of initial presentation. Two patients had negative coccidioidal screening tests upon admission but later seroconverted. All patients had extrapulmonary involvement, and all recovered after appropriate treatment. A review of the literature reveals that the presentations and outcomes of neonatal coccidioidomycosis vary widely. CONCLUSIONS: There is significant variability in the presentation of coccidioidomycosis in the neonatal period, and diagnosis may be challenging. In endemic regions, healthcare providers should consider coccidioidomycosis in their differential diagnoses of ill-appearing neonates that do not respond to treatment.

Update on CVD 103-HgR single-dose, live oral cholera vaccine.

Cholera remains endemic in >50 countries, putting millions at risk, especially young children for whom killed vaccines offer limited protection. An oral, live attenuated vaccine - CVD 103-HgR (Vaxchora vaccine) - was licensed by the US FDA in 2016 for adults aged 18-64 years traveling to endemic regions, based on clinical trials in human volunteers showing the vaccine was well tolerated and conferred 90% efficacy within 10 days. The evidence base for Vaxchora vaccine has expanded with additional clinical trial data, in older adults (aged 46-64 years) and children (aged 2-17 years), demonstrating that the vaccine produces a strong vibriocidal antibody response. Over 68,000 doses have been administered in the United States, with no new safety signals. The dose volume has been reduced in children to improve acceptability, and cold chain requirements are less st ringent, at +2°C─+8°C. The vaccine has recently been licensed in the Untied States for children aged 2-17 years, in Europe for individuals aged ≥2 years, and for home administration in Europe. Next steps include a Phase 4 study in infants (6-23 months). Additional information is needed regarding duration of immunity, the need for and timing of revaccination, and efficacy data from lower-middle-income countries.

Congenital Coccidioidomycosis: A Case Report and Review of the Literature.

Coccidioidomycosis in neonates is rare and the acquisition of disease in this age group is not well understood. Congenital coccidioidomycosis is very rare, usually associated with coccidioidal placentitis. Only a handful of cases of congenital coccidioidomycosis have been described in the literature. We describe an infant with congenital coccidioidomycosis delivered by cesarean section to a mother who was diagnosed with disseminated disease in the second trimester and summarize the available literature on congenital coccidioidomycosis.

Insight Into Seeded Tau Fibril Growth From Molecular Dynamics Simulation of the Alzheimer's Disease Protofibril Core.

Aggregates of the microtubule associated tau protein are a major constituent of neurofibrillary lesions that define Alzheimer's disease (AD) pathology. Increasing experimental evidence suggests that the spread of tau neurofibrillary tangles results from a prion-like seeding mechanism in which small oligomeric tau fibrils template the conversion of native, intrinsically disordered, tau proteins into their pathological form. By using atomistic molecular dynamics (MD) simulations, we investigate the stability and dissociation thermodynamics of high-resolution cryo-electron microscopy (cryo-EM) structures of both the AD paired-helical filament (PHF) and straight filament (SF). Non-equilibrium steered MD (SMD) center-of-mass pulling simulations are used to probe the stability of the protofibril structure and identify intermolecular contacts that must be broken before a single tau peptide can dissociate from the protofibril end. Using a combination of exploratory metadynamics and umbrella sampling, we investigate the complete dissociation pathway and compute a free energy profile for the dissociation of a single tau peptide from the fibril end. Different features of the free energy surface between the PHF and SF protofibril result from a different mechanism of tau unfolding. Comparison of wild-type tau PHF and post-translationally modified pSer356 tau shows that phosphorylation at this site changes the dissociation free energy surface of the terminal peptide. These results demonstrate how different protofibril morphologies template the folding of endogenous tau in distinct ways, and how post-translational modification can perturb the folding mechanism.

Hydrocephalus in Mexican children with Coccidioidal Meningitis: Clinical, serological, and neuroimaging findings.

BACKGROUND: Coccidioidal meningitis (CM) is a fungal infectious disease that rarely affects children. Even in endemic areas, coccidiomycosis rarely affects the pediatric population. However, 40% of affected children develop hydrocephalus. Here, we describe the clinical, serological, and neuroimaging findings in a series of Mexican children admitted to our neurosurgical service with hydrocephalus and subsequently diagnosed with CM. METHODS: We report a prospective series of pediatric patients with hydrocephalus secondary to CM in an endemic area at the north of Mexico. Our report includes children with CM who were hospitalized from 2015 to 2019 in a regional hospital in Torreón, Coahuila. Clinical evolution was monitored for 1 year after hospital discharge. RESULTS: Our series include five children with CM (2-17-years-old, three female), who were hospitalized for hydrocephalus and developed intracranial hypertension. The most frequent neuroimaging findings were leptomeningeal enhancement (5/5) and basal arachnoiditis (4/5), followed by asymmetric hydrocephalus (3/5), abnormalities in fourth ventricle morphology (3/5), and cerebral vasculitis (2/5). CM was diagnosed by positive serology or pathology studies. All children were initially managed with fluconazole and a shunt was placed for management of hydrocephalus. Four patients recovered without permanent neurological deficits and one subject developed persistent vegetative state. One year after hospital discharge, none of the subjects died. CONCLUSION: This series contributes to the limited number of pediatric CM cases reported in the literature, and describes neuroimaging findings in the pediatric population. The cases here presented show that the identification of Coccidioides as causal agent in pediatric meningitis is crucial for targeted treatment and can affect dramatically neurological prognosis. Furthermore, our report stresses that even in endemic areas pediatric coccidiomycosis represents a diagnostic challenge, which is further exacerbated by the limited availability of resources in these regions. Therefore, a positive immunoglobulin G by enzyme immunoassay is enough for diagnosis of CM in endemic areas without access to CF.

Long-Term Immunogenicity of Live Oral Cholera Vaccine CVD 103-HgR in Adolescents Aged 12-17 Years in the United States.

As part of a phase 4, randomized, double-blind, placebo-controlled trial to assess the immunogenicity and safety of PXVX0200 in children and adolescents aged 2-17 years, a subset of 73 adolescent subjects aged 12-17 years was followed for 2 years after vaccination and had blood collected for antibody assays on days 1, 11, 29, 91, 181, 365, 547, and 730. Endpoints included serum vibriocidal antibody (SVA) seroconversion, defined as a 4-fold or greater rise in antibody titer over baseline; geometric mean titers (GMTs); and geometric mean fold increase (GMFI) over baseline. Serum vibriocidal antibody seroconversion persisted in most subjects, with a rate of 64.5% noted at day 730. Geometric mean titers and GMFI both peaked at day 11 and remained greater than baseline at all time points, including day 730. Vaccination with PXVX0200 produces an immune response which persists for at least 2 years in adolescents aged 12-17 years.

Amyloid Oligomers: A Joint Experimental/Computational Perspective on Alzheimer's Disease, Parkinson's Disease, Type II Diabetes, and Amyotrophic Lateral Sclerosis.

Protein misfolding and aggregation is observed in many amyloidogenic diseases affecting either the central nervous system or a variety of peripheral tissues. Structural and dynamic characterization of all species along the pathways from monomers to fibrils is challenging by experimental and computational means because they involve intrinsically disordered proteins in most diseases. Yet understanding how amyloid species become toxic is the challenge in developing a treatment for these diseases. Here we review what computer, in vitro, in vivo, and pharmacological experiments tell us about the accumulation and deposition of the oligomers of the (Aß, tau), α-synuclein, IAPP, and superoxide dismutase 1 proteins, which have been the mainstream concept underlying Alzheimer's disease (AD), Parkinson's disease (PD), type II diabetes (T2D), and amyotrophic lateral sclerosis (ALS) research, respectively, for many years.

Isavuconazole as Salvage Therapy for Refractory Pediatric Coccidioidal Meningitis.

Coccidioidal meningitis remains difficult to treat. The newer triazole, isavuconazole, has demonstrated efficacy in invasive fungal disease with less side effects than other azoles. We describe a case of refractory pediatric coccidioidal meningitis with disease stabilization and improvement on isavuconazole after failing treatment with other antifungal agents.

Safety and Immunogenicity of Live Oral Cholera Vaccine CVD 103-HgR in Children Aged 2-5 Years in the United States.

In a phase 4, randomized, placebo-controlled, double-blind, multicenter study, to assess the safety and immunogenicity of live, attenuated cholera vaccine PXVX0200 in children aged 2-5 years in the United States, 172 volunteers were randomized 6:1 to receive a single dose of 1 × 109 colony forming units (CFU) of PXVX0200 or placebo. Immunogenicity endpoints included serum vibriocidal antibody (SVA) levels on days 1, 11, and 29. Safety was assessed by comparing solicited signs and symptoms on days 1-8, unsolicited adverse events through day 29, and serious adverse events (SAEs) through day 181. The SVA seroconversion rates 10 days after immunization were 98.1% and 0% in vaccine and placebo recipients, respectively, and the vaccine seroconversion rate was non-inferior to the 93.5% rate seen in the bridging population of adults aged 18-45 years from a lot consistency study. Most reactogenicity was mild to moderate, and there were no study-related SAEs. PXVX0200 appears safe and immunogenic in children aged 2-5 years.

The proline-rich domain promotes Tau liquid-liquid phase separation in cells.

Tau protein in vitro can undergo liquid-liquid phase separation (LLPS); however, observations of this phase transition in living cells are limited. To investigate protein state transitions in living cells, we attached Cry2 to Tau and studied the contribution of each domain that drives the Tau cluster in living cells. Surprisingly, the proline-rich domain (PRD), not the microtubule binding domain (MTBD), drives LLPS and does so under the control of its phosphorylation state. Readily observable, PRD-derived cytoplasmic condensates underwent fusion and fluorescence recovery after photobleaching consistent with the PRD LLPS in vitro. Simulations demonstrated that the charge properties of the PRD predicted phase separation. Tau PRD formed heterotypic condensates with EB1, a regulator of plus-end microtubule dynamic instability. The specific domain properties of the MTBD and PRD serve distinct but mutually complementary roles that use LLPS in a cellular context to implement emergent functionalities that scale their relationship from binding α-beta tubulin heterodimers to the larger proportions of microtubules.

Case Study of Phrenic Nerve Paralysis: "I Can't Breathe!"

BACKGROUND: The anatomic course of the phrenic nerve runs in the fascia covering the anterior scalene muscle. Interscalene blocks are commonly performed by an anesthesiologist for shoulder surgery, such as a rotator cuff repair, total shoulder replacement, humeral fracture, or other arm surgery. Phrenic nerve palsy or paralysis is a known complication from interscalene block and is covered in multiple case reports and series in both Anesthesia and Neurosurgical literature, but only one case report in the Emergency Medicine literature. CASE REPORT: This case involves a 57-year-old man who had an uncomplicated arthroscopic rotator cuff repair with placement of interscalene block under care of anesthesia. He was discharged with a pain pump in place and then subsequently presented to the Emergency Department (ED) later that same day for evaluation of dyspnea. Using point-of-care ultrasound, his right diaphragm did not appear to be moving. Chest x-ray study revealed an elevated right hemidiaphragm. He was diagnosed with iatrogenic right phrenic nerve paralysis from interscalene block. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Emergent diagnosis of phrenic nerve paralysis in the ED is complicated by a distressed patient and need for quick intervention. Most formal tests for this diagnosis are not immediately available to emergency physicians. Ultrasound is a rapid and reproducible, noninvasive resource with high sensitivity and specificity, making it an ideal imaging modality for the emergent evaluation of possible phrenic nerve palsy or paralysis.