Role of Cardiac Anesthesiologists in Intraoperative Enhanced Recovery After Cardiac Surgery (ERACS) Protocol: A Retrospective Single-Center Study Analyzing Preliminary Results of a Yearlong ERACS Protocol Implementation.

INTRODUCTION: Enhanced recovery after cardiac surgery (ERACS) has been gaining rapid acceptance after multiple studies have demonstrated promising results in improved outcomes of enhanced recovery after surgery in other surgical fields (eg, colorectal, orthopedic, thoracic, etc). Cardiac surgery has several unique challenges, including sternotomy, cardiopulmonary bypass and associated coagulopathy, blood transfusion, and postoperative intensive care requirement. Nonetheless, selective cardiac surgical patients can still benefit from ERACS. Guidelines for perioperative care in cardiac surgery, previously published by the ERACS Society, are weighted heavily in preoperative and postoperative management without much focus on intraoperative care provided by anesthesiologists. To address this gap and to explore anesthesiology's contribution in achieving ERACS, the study authors' cardiac anesthesiology division, in collaboration with cardiac surgery, introduced the ERACS protocol in their institution in February 2020. METHODS: The cardiac anesthesiology division, in collaboration with cardiac surgery, introduced the ERACS protocol consisting of multimodal opioid-sparing analgesia, including the introduction of regional blocks, hemostasis management protocol, reversal of neuromuscular blockade, and administration of antiemetics in the authors' institution in February 2020. They have conducted a retrospective chart review study comparing patients who have received ERACS measures with a similar historic cohort who underwent cardiac surgery prior to initiation of an ERACS protocol. The primary outcomes of the study were to determine patients' time to extubation, postoperative opioid consumption, intensive care unit (ICU) length of stay (LOS), and incidence of postoperative complications (eg, postoperative nausea vomiting [PONV], bleeding, ICU readmission, delirium. RESULTS: The ERACS patients showed reduced opioid consumption (intraoperative fentanyl; postoperative fentanyl, as well as oxycodone, in the first 6 hours postoperatively), lesser mechanical ventilation (2.5 hours less), shorter ICU stays (5 hours less), shorter hospital LOS (1 day), and lesser incidence of PONV. None of the ERACS patients required blood transfusion. The study authors performed an anonymous survey among the anesthesiologists and ICU providers to assess providers' satisfaction, which showed 92% of survey takers agreed that the ERACS protocol should be continued for future cardiac patients, and 61% of survey takers reported superior pain control in ERACS group of patients while managing those patients. DISCUSSION: The ERACS is achievable after the careful implementation of a series of measures. It does not signify only fast-track extubation and opioid-sparing analgesia, and must be implemented in the entire perioperative period beginning from preoperative clinic to postoperative rehabilitation. Cardiac anesthesiologists play a vital role in execution of intraoperative ERACS measures. Both providers and patients themselves are key stakeholders. A larger randomized prospective trial is warranted to solidify the inference.

An Intraoperative Episode of Severe Type II Renal Tubular Acidosis in an 83-Year-Old Woman.

Type II renal tubular acidosis (RTA) is a rare defect in bicarbonate transport that can cause serious metabolic derangements. We report the case of a spontaneous, isolated intraoperative episode of severe type II RTA in an elderly woman who presented for radical neck dissection, mandible excision, and flap creation. Intraoperatively, she developed a stark metabolic acidosis with hypokalemia, progressively worsening base excess, and prolific urine output. Aggressive resuscitation with bicarbonate corrected all metabolic abnormalities. There was no identifiable trigger and the patient was successfully discharged with no further recurrences during hospitalization. The inability to identify the clinical presentation of RTA perioperatively can lead to poor outcomes.

A Retrospective Analysis of Respiratory Complications under General Anesthesia during EBUS-TBNA.

Background: EBUS-TBNA is an established technique for diagnostically sampling intrathoracic masses and lymph nodes. While the procedure is commonly conducted under general anesthesia (GA), little is known regarding the association between anesthetic management and perioperative respiratory complications. Here, we aim to evaluate this association among patients presenting for EBUS-TBNA. Methods: 586 patients receiving GA for EBUS-TBNA between 2012 and 2018 were retrospectively evaluated. The primary endpoint was the occurrence of perioperative respiratory complications and the secondary endpoint was procedure end to OR exit time (minutes). Respiratory complications were defined as episodes of severe (SpO2 <85%) or prolonged (SpO2<90% for >5 min) hypoxemia, bronchospasm, and postoperative ventilation that could not be directly attributed to procedural invasiveness. Results: Among all patients, 79 (13.5%) had respiratory complications. Four patient characteristics were associated with respiratory complications: home oxygen use (OR 2.39; 95% CI 1.26-4.45; P = 0.007), pre-existing respiratory disease (OR 2.01; CI 1.21-3.29; P = 0.005), ASA class (P = 0.03), and albuterol administration intra-operatively (OR 2.22; CI 1.23-3.92; P = 0.007). No anesthetic factors were found to be statistically significant. Procedures with respiratory complications had a longer duration (mean time 88.7 min vs. 111.8 min; P = 0.00009), prolonged time to extubation (mean time 11.9 min vs. 14.2 min; P = 0.039), and stayed in the room longer after extubation (mean time 18.4 min vs. 23.1 min; P = 0.0016). When comparing types of GA, there were no significant differences between volatile anesthetics versus TIVA (12.7% vs. 14.6%, P = 0.54). Conclusions: Pre-existing patient characteristics, as opposed to anesthetic factors, are associated with respiratory complications during EBUS-TBNA.

The Good, Bad, and Ugly of Inferior Vena Cava (IVC) Filters: Anesthetic Management for IVC Filter Retrieval in the Setting of Filter Thrombosis.

We describe a case of a 58-year-old man presenting to the interventional radiology (IR) suite for inferior vena cava (IVC) filter retrieval and potential intravascular iliocaval stent reconstruction in the setting of anticoagulation and uncontrolled hypertension. This patient had recently undergone iliocaval thrombectomy with IVC venoplasty four weeks prior to presentation. Induction of anesthesia and endotracheal intubation occurred without complication. The patient received two large-bore intravenous (IV) catheters and a radial artery catheter for hemodynamic monitoring. Blood was cross-matched and kept in the IR suite, anticipating bleeding from a potential injury to the IVC during filter retrieval. Fortunately, the thrombosed filter was removed without complication. This case illustrates the importance of in-depth anesthetic planning for so-called "benign" surgical procedures and highlights the challenges faced in non-operating room locations for anesthesiologists.

Copper deficiency myeloneuropathy with a history of malabsorption: a tale of two cases.

Acquired copper deficiency is rare but often seen among patients with intestinal malabsorption syndromes. Often times, these patients can develop myeloneuropathies with copper deficiency as an elusive culprit. With early diagnosis and appropriate supplementation, many symptoms, such as myeloneuropathy, can be reversed entirely. Checking copper levels should be included in the workup of myeloneuropathies to prevent irreversible damage and improving morbidity and mortality.

A novel early mobility bundle improves length of stay and rates of readmission among hospitalized general medicine patients.

Inpatient early mobility initiatives are effective therapeutic interventions for improving patient outcomes and decreasing use of hospital resources among adult ICU and general medicine patients. To establish and demonstrate guidelines for early patient ambulation, we developed and implemented a novel multidisciplinary mobility bundle utilizing the JH-HLM (Johns Hopkins Highest Level of Mobility) scale for mobility classification, on a single adult general medicine unit of a community hospital. Our results show that patients admitted to the unit after implementation of the mobility bundle had improved mobility scores, reduced rates of 30-day hospital readmission, and a shortened length of hospital stay. This study emphasizes the importance of measuring mobility using a systematic method, easing workflow among unit practitioners, and allowing mobility initiatives to be jointly driven by nursing, physical therapy, and physicians.

A 33-Year-Old Man With Shortness of Breath, Leukocytosis, and Intermittent Fevers.

CASE PRESENTATION: A 33-year-old man with ulcerative colitis (UC) and primary sclerosing cholangitis presented with worsening shortness of breath, nonproductive cough, and intermittent fevers after he was found to have a WBC count of 27,000 cells/µL on an outpatient laboratory evaluation. He reported feeling progressively unwell with intermittent right upper quadrant pain and shortness of breath since a hospital admission for a UC flare 6 months prior, during which he was first diagnosed with primary sclerosing cholangitis. He noted that prior to that admission 6 months ago, his UC had been in remission for > 10 years. He reported fevers up to 38.9°C on and off for several weeks but was afebrile (37.2°C) on presentation. He endorsed non-bloody diarrhea, chills, night sweats, leg swelling, and associated leg pain. He had a cough but denied sputum production. He reported no recent travels and denied sick contacts. His medications included mesalamine, ursodiol, montelukast, and an albuterol inhaler.

Multi-gene technical assessment of qPCR and NanoString n-Counter analysis platforms in cynomolgus monkey cardiac allograft recipients.

Quantitative gene expression profiling of cardiac allografts characterizes the phenotype of the alloimmune response, yields information regarding differential effects that may be associated with various anti-rejection drug regimens, and generates testable hypotheses regarding the pathogenesis of the chronic rejection lesions typically observed in non-human primate heart transplant models. The goal of this study was to assess interplatform performance and variability between the relatively novel NanoString nCounter Analysis System, ΔΔCT (relative) RT-qPCR, and standard curve (absolute) RT-qPCR utilizing cynomolgus monkey cardiac allografts. Methods for RNA isolation and preamplification were also systematically evaluated and effective methods are proposed. In this study, we demonstrate strong correlation between the two RT-qPCR methods, but variable and, at times, weak correlation between RT-qPCR and NanoString. NanoString fold change results demonstrate less sensitivity to small changes in gene expression than RT-qPCR. These findings appear to be driven by technical aspects of each platform that influence the conditions under which each technique is ideal. Collectively, our data contribute to the general effort to optimally utilize gene expression profiling techniques, not only for transplanted tissues, but for many other applications where accurate rank-order of gene expression versus precise quantification of absolute gene transcript number may be relatively valuable.

The CFTR Corrector, VX-809 (Lumacaftor), Rescues ABCA4 Trafficking Mutants: a Potential Treatment for Stargardt Disease.

BACKGROUND/AIMS: Mutations in ABCA4 cause Stargardt macular degeneration, which invariably ends in legal blindness. We studied two common mutants, A1038V (in NBD1) and G1961E (in NBD2), with the purpose of exploring how they interact with the cell's quality control mechanism. The study was designed to determine how these mutants can be rescued. METHODS: We expressed wt and mutant ABCA4 in HEK293 cells and studied the effect of the mutations on trafficking and processing and the ability of correctors to rescue them. We used a combination of western blotting, confocal microscopy and surface biotinylation coupled with pulldown of plasma membrane proteins. RESULTS: G1961E is sensitive to inhibitors of the aggresome, tubacin and the lysosome, bafilomycin A. Both mutants cause a reduction in heat shock protein, Hsp27. Incubation of HEK293 cells expressing the mutants with VX-809, an FDA approved drug for the treatment of cystic fibrosis, increased the levels of A1038V and G1961E by 2- to 3-fold. Importantly, VX-809 increased the levels of both mutants at the plasma membrane suggesting that trafficking had been restored. Transfecting additional Hsp27 to the cells also increased the steady state levels of both mutants. However, in combination with VX-809 the addition of Hsp27 caused a dramatic increase in the protein expression particularly in the G1961 mutant which increased approximately 5-fold. CONCLUSION: Our results provide a new mechanism for the rescue of ABCA4 trafficking mutants based on the restoration of Hsp27. Our results provide a pathway for the treatment of Stargardt disease.

Restoration of F508-del Function by Transcomplementation: The Partners Meet in the Endoplasmic Reticulum.

BACKGROUND/AIMS: Because of the small size of adeno-associated virus, AAV, the cystic fibrosis conductance regulator, CFTR, cDNA is too large to fit within AAV and must be truncated. We report here on two truncated versions of CFTR, which, when inserted into AAV1 and used to infect airway cells, rescue F508-del CFTR via transcomplementation. The purpose of this study is to shed light on where in the cell transcomplementation occurs and how it results in close association between the endogenous F508-del and truncated CFTR. METHODS: We treated CF airway cells (CFBE41o-) with AAV2/1 (AAV2 inverted terminal repeats/AAV1 capsid) containing truncated forms of CFTR, ∆264 and ∆27-264 CFTR, who can restore the function of F508-del by transcomplementation. We addressed the aims of the study using a combination of confocal microscopy and short circuit currents measurements. For the latter, CF bronchial epithelial cells (CFBE) were grown on permeable supports. RESULTS: We show that both F508del and the truncation mutants colocalize in the ER and that both the rescued F508-del and the transcomplementing mutants reach the plasma membrane together. There was significant fluorescence resonance energy transfer (FRET) between F508-del and the transcomplementing mutants within the endoplasmic reticulum (ER), suggesting that transcomplementation occurs through a bimolecular interaction. We found that transcomplementation could increase the Isc in CFBE41o- cells stably expressing additional wt-CFTR or F508-del and in parental CFBE41o- cells expressing endogenous levels of F508-del. CONCLUSION: We conclude that the functional rescue of F508-del by transcomplementation occurs via a bimolecular interaction that most likely begins in the ER and continues at the plasma membrane. These results come at an opportune time for developing a gene therapy for CF and offer new treatment options for a wide range of CF patients.

Rescue of CFTR NBD2 mutants N1303K and S1235R is influenced by the functioning of the autophagosome.

The missing phenylalanine at position 508, located in nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane regulator (CFTR), is the most common cystic fibrosis mutation. Severe disease-causing mutations also occur in NBD2. To provide information on potential therapeutic strategies for mutations in NBD2, we used a combination of biochemical, cell biological and electrophysiological approaches and newly created cell lines to study two disease-causing NBD2 mutants, N1303K and S1235R. We observed that neither was sensitive to E64, a cysteine protease inhibitor. However, further investigation showed that when treated with a combination of correctors, C4 + C18, both mutants also responded to E64. Further exploration to assess aggresome throughput using the autophagy regulator LC3 as a marker showed that, in the absence of correctors, N1303K showed a stalled throughput of LC3-II to the aggresome. The throughput became active again after treatment with the corrector combination C4 + C18. Confocal microscopic studies showed that the N1303K and S1235R mutant proteins both co-localized with LC3, but this co-localization was abolished by the corrector combination and, to a lesser extent, by VX-809. Both the corrector combination and VX-809 increased the CFTR chloride channel function of both mutants. We conclude that correctors have a dual effect, particularly on N1303K: they improve trafficking and function at the plasma membrane and reduce the association with autophagosomes. After treatment with correctors persistent degradation by the autophagosome may limit restoration of function. Thus, mutations in NBD2 of CFTR, in contrast to ΔF508-CFTR, may require additional personalized strategies to rescue them.

The CFTR-Associated Ligand Arrests the Trafficking of the Mutant ΔF508 CFTR Channel in the ER Contributing to Cystic Fibrosis.

BACKGROUND/AIMS: The CFTR-Associated Ligand (CAL), a PDZ domain containing protein with two coiled-coil domains, reduces cell surface WT CFTR through degradation in the lysosome by a well-characterized mechanism. However, CAL's regulatory effect on ΔF508 CFTR has remained almost entirely uninvestigated. METHODS: In this study, we describe a previously unknown pathway for CAL by which it regulates the membrane expression of ΔF508 CFTR through arrest of ΔF508 CFTR trafficking in the endoplasmic reticulum (ER) using a combination of cell biology, biochemistry and electrophysiology. RESULTS: We demonstrate that CAL is an ER localized protein that binds to ΔF508 CFTR and is degraded in the 26S proteasome. When CAL is inhibited, ΔF508 CFTR retention in the ER decreases and cell surface expression of mature functional ΔF508 CFTR is observed alongside of enhanced expression of plasma membrane scaffolding protein NHERF1. Chaperone proteins regulate this novel process, and ΔF508 CFTR binding to HSP40, HSP90, HSP70, VCP, and Aha1 changes to improve ΔF508 CFTR cell surface trafficking. CONCLUSION: Our results reveal a pathway in which CAL regulates the cell surface availability and intracellular retention of ΔF508 CFTR.

Selective CD28 Inhibition Modulates Alloimmunity and Cardiac Allograft Vasculopathy in Anti-CD154-Treated Monkeys.

BACKGROUND: Selective CD28 inhibition is actively pursued as an alternative to B7 blockade using cytotoxic T lymphocyte antigen 4 Ig based on the hypothesis that the checkpoint immune regulators cytotoxic T lymphocyte antigen 4 and programmed death ligand 1 will induce tolerogenic immune signals. We previously showed that blocking CD28 using a monovalent nonactivating reagent (single-chain anti-CD28 Fv fragment linked to alpha-1 antitrypsin [sc28AT]) synergizes with calcineurin inhibitors in nonhuman primate (NHP) kidney and heart transplantation. Here, we explored the efficacy of combining a 3-week "induction" sc28AT treatment with prolonged CD154 blockade. METHODS: Cynomolgus monkey heterotopic cardiac allograft recipients received sc28AT (10 mg/kg, d0-20, n = 3), hu5C8 (10-30 mg/kg, d0-84, n = 4), or combination (n = 6). Graft survival was monitored by telemetry. Protocol biopsies and graft explants were analyzed for International Society of Heart and Lung Transplantation acute rejection grade and cardiac allograft vasculopathy score. Alloantibody, T-cell phenotype and regulatory T cells were analyzed by flow cytometry. Immunochemistry and gene expression (NanoString) characterized intra-graft cellular infiltration. RESULTS: Relative to modest prolongation of median graft survival time with sc28AT alone (34 days), hu5C8 (133 days), and sc28AT + hu5C8 (141 days) prolonged survival to a similar extent. CD28 blockade at induction, added to hu5C8, significantly attenuated the severity of acute rejection and cardiac allograft vasculopathy during the first 3 months after transplantation relative to hu5C8 alone. These findings were associated with decreased proportions of circulating CD8 and CD3CD28 T cells, and modulation of inflammatory gene expression within allografts. CONCLUSIONS: Induction with sc28AT promotes early cardiac allograft protection in hu5C8-treated NHPs. These results support further investigation of prolonged selective CD28 inhibition with CD40/CD154 blockade in NHP transplants.

SOX2 expression is an early event in a murine model of EGFR mutant lung cancer and promotes proliferation of a subset of EGFR mutant lung adenocarcinoma cell lines.

OBJECTIVES: Primary and acquired resistance to EGFR TKIs in EGFR mutant lung cancer occurs primarily through secondary mutations in EGFR or Met amplification. Drug resistance can also be mediated by expression of pluripotency transcription factors, such as OCT4, SOX2 and NANOG that decrease terminal differentiation. In this study, we investigated the expression and role of SOX2 in model systems of EGFR mutant tumors. MATERIALS AND METHODS: Immunoblotting or immunohistochemistry was used to assess expression of pluripotency transcription factors in lungs of transgenic mice or in human NSCLC cell lines. Expression of SOX2 was reduced by shRNA knockdown, and response to erlotinib and cellular proliferation were assessed. RESULTS AND CONCLUSION: Induction of mutant EGFR in transgenic CCSP-rtTA/TetO-EGFR(L858R/T790M) mice correlated with increased OCT4 and SOX2 expression in lung tissue prior to tumor development. Established lung tumors retained SOX2 expression. To assess a role for SOX2 in tumorigenesis, a panel of NSCLC cell lines with activating EGFR mutations was assessed for SOX2 expression. Two of six cell lines with mutant EGFR showed detectable SOX2 levels, suggesting SOX2 expression did not correlate with EGFR mutation status. To assess the role of SOX2 in these cell lines, HCC827 and H1975 cells were infected with lentivirus containing SOX2 shRNA. Knockdown of SOX2 decreased proliferation in both cell lines and increased sensitivity to erlotinib in HCC827 cells. Because constitutive activation of the PI3K/Akt pathway is associated with EGFR TKI resistance, cells were treated with PI3K/AKT inhibitors and expression of SOX2 was examined. PI3K/Akt inhibitors decreased SOX2 expression in a time-dependent manner. These data suggest targeting SOX2 may provide therapeutic benefit in the subset of EGFR-mutant tumors with high constitutive levels of SOX2, and that until more direct means of inhibiting SOX2 are developed, PI3K/Akt inhibitors might be useful to inhibit SOX2 in EGFR TKI resistant tumors.

Toxoplasma gondii salvages sphingolipids from the host Golgi through the rerouting of selected Rab vesicles to the parasitophorous vacuole.

The obligate intracellular protozoan Toxoplasma gondii actively invades mammalian cells and, upon entry, forms its own membrane-bound compartment, named the parasitophorous vacuole (PV). Within the PV, the parasite replicates and scavenges nutrients, including lipids, from host organelles. Although T. gondii can synthesize sphingolipids de novo, it also scavenges these lipids from the host Golgi. How the parasite obtains sphingolipids from the Golgi remains unclear, as the PV avoids fusion with host organelles. In this study, we explore the host Golgi-PV interaction and evaluate the importance of host-derived sphingolipids for parasite growth. We demonstrate that the PV preferentially localizes near the host Golgi early during infection and remains closely associated with this organelle throughout infection. The parasite subverts the structure of the host Golgi, resulting in its fragmentation into numerous ministacks, which surround the PV, and hijacks host Golgi-derived vesicles within the PV. These vesicles, marked with Rab14, Rab30, or Rab43, colocalize with host-derived sphingolipids in the vacuolar space. Scavenged sphingolipids contribute to parasite replication since alterations in host sphingolipid metabolism are detrimental for the parasite's growth. Thus our results reveal that T. gondii relies on host-derived sphingolipids for its development and scavenges these lipids via Golgi-derived vesicles.

Comprehensive genomic analysis identifies SOX2 as a frequently amplified gene in small-cell lung cancer.

Small-cell lung cancer (SCLC) is an exceptionally aggressive disease with poor prognosis. Here, we obtained exome, transcriptome and copy-number alteration data from approximately 53 samples consisting of 36 primary human SCLC and normal tissue pairs and 17 matched SCLC and lymphoblastoid cell lines. We also obtained data for 4 primary tumors and 23 SCLC cell lines. We identified 22 significantly mutated genes in SCLC, including genes encoding kinases, G protein-coupled receptors and chromatin-modifying proteins. We found that several members of the SOX family of genes were mutated in SCLC. We also found SOX2 amplification in ∼27% of the samples. Suppression of SOX2 using shRNAs blocked proliferation of SOX2-amplified SCLC lines. RNA sequencing identified multiple fusion transcripts and a recurrent RLF-MYCL1 fusion. Silencing of MYCL1 in SCLC cell lines that had the RLF-MYCL1 fusion decreased cell proliferation. These data provide an in-depth view of the spectrum of genomic alterations in SCLC and identify several potential targets for therapeutic intervention.

Seizure tests distinguish intermittent fasting from the ketogenic diet.

PURPOSE: Calorie restriction can be anticonvulsant in animal models. The ketogenic diet was designed to mimic calorie restriction and has been assumed to work by the same mechanisms. We challenged this assumption by profiling the effects of these dietary regimens in mice subjected to a battery of acute seizure tests. METHODS: Juvenile male NIH Swiss mice received ketogenic diet or a normal diet fed in restricted quantities (continuously or intermittently) for ∼12 days, starting at 3-4 weeks of age. Seizures were induced by the 6 Hz test, kainic acid, maximal electroshock, or pentylenetetrazol. RESULTS: The ketogenic and calorie-restricted diets often had opposite effects depending on the seizure test. The ketogenic diet protected from 6 Hz-induced seizures, whereas calorie restriction (daily and intermittent) increased seizure activity. Conversely, calorie restriction protected juvenile mice against seizures induced by kainic acid, whereas the ketogenic diet failed to protect. Intermittent caloric restriction worsened seizures induced by maximal electroshock but had no effect on those induced by pentylenetetrazol. DISCUSSION: In contrast to a longstanding hypothesis, calorie restriction and the ketogenic diet differ in their acute seizure test profiles, suggesting that they have different underlying anticonvulsant mechanisms. These findings highlight the importance of the 6 Hz test and its ability to reflect the benefits of ketosis and fat consumption.