Function and interaction of coronavirus ion channel proteins

Objective: COVID-19 has emerged as a significant global health crisis causing devastating effects on world population accounting for over 6 million deaths worldwide. Although acute RTI is the prevalent cause of morbidity, kidney outcomes centered on a spectrum of AKI have evolved over the course of the pandemic. Especially the emerging variants have posed a daunting challenge to the scientific communities, prompting an urging requirement for global contributions in understanding the viral dynamics. In addition to canonical genes, several subgroup- specific accessory genes are located between the S and E genes of coronaviruses regarding which little is known. Previous studies have shown that accessory proteins (aps) in viruses function as viroporins that regulate viral infection, propagation and egress [1]. In this study we attempted to characterize the function of aps of coronavirus variants as ion channels. Furthermore, we also probed the interaction of ap4 with the host system. Method(s): Serial passaging (selection pressure), growth kinetics, confocal imaging, genome sequence analysis and proteomics were performed in Huh-7, MRC5 cells and/or human monocyte derived macrophages. Potassium uptake assay was performed in a Saccharo myces cerevisiae strain, which lacks the potassium transporters trk1 and trk2. Ion conductivity experiments were performed in Xenopus laevis oocytes using Two Electrode Voltage Clamp (TEVC) method. Result(s): Serial passaging demonstrated the acquisition of several frameshift mutations in ORF4 resulting in C-terminally truncated protein versions (ap4 and ap4a) and indicate a strong selection pressure against retaining a complete ORF4 in vitro. Growth kinetics in primary cells illustrated a reduction of viral titers when the full-length ap4 was expressed compared to the C-terminally truncated protein ap4a. Confocal imaging showed that ap4 and ap4a are not exclusively located in a single cellular compartment. Potassium uptake assay in yeast and TEVC analyses in Xenopus oocytes showed that ap4 and ap4a act as a weak K+ selective ion channel. In addition, accessory proteins of other virus variants also elicited microampere range of currents. Conclusion(s): Our study provides the first evidence that ap4 and other accessory proteins of coronavirus variants act as viroporins. Future studies are aimed at demonstrating the role of ap4 during the viral life cycle by modulating ion homeostasis of host cell in vivo (interacting proteins obtained from proteomic studies) and thereby serve as a tool for potential drug target.

Pathogenesis of encephalopathy and delirium: neocortical neuronal membrane potential and action potential firing are changed by coronavirus-associated cytokines

Introduction: Encephalopathy and delirium are common following coronavirus infection [1], and the associated neuroinflammation often results in long-term behavioral and cognitive impairment. Neurovirulent cytokines (NVC) are strongly implicated in the pathogenesis of coronavirus encephalopathy [2]. We hypothesized that characterizing the abnormal signaling in NVC exposed neurons will enable us to identify targets to treat encephalopathy and prevent its downstream effects. Method(s): We incubated primary mouse neocortical cultures in NVC known to be increased in coronavirus encephalopathy (TNF-alpha, IL-1beta, IL-6, IL-12 and IL-15). Using whole-cell patch clamp methods, we tested how neuronal function was impacted by 22-28-h exposure to NVC. Result(s): We found that NVC depolarized the resting membrane potential (RMP), reduced the firing threshold of neocortical neurons, and increased baseline spontaneous action potential (AP) firing. NVC altered the sensitivity (or input-output properties) of single neurons to changes in their microenvironment. Specifically, decreasing external Ca2+ and Mg2+ from physiological to low (1.1-0.2 mM) levels increased evoked AP firing in control, but not following exposure to NVC. AP firing threshold and spontaneous firing rates returned to control levels 1 h after NVC wash-out. However, the RMP and attenuated sensitivity of evoked APs to changes in the microenvironment remained persistently abnormal suggesting two distinct mechanisms were at play. Interestingly, hyperpolarizing the RMP reversed this altered response. Conclusion(s): Sustained exposure to NVC reversibly depolarizes neocortical neuronal RMP, altering excitability and the ability of neurons to respond to microenvironment changes. By characterizing the pathogenesis of the underlying changes in neuronal function in our model of coronavirus encephalopathy we will identify intervenable drug targets.

Primary hyperparathyroidism in young patients is associated with metabolic disorders: a prospective comparative study.

BACKGROUND: Components of metabolic syndrome can be observed in patients with primary hyperparathyroidism (PHPT). The link between these disorders remains unclear due to the lack of relevant experimental models and the heterogeneity of examined groups. The effect of surgery on metabolic abnormalities is also controversial. We conducted a comprehensive assessment of metabolic parameters in young patients with PHPT. METHODS: One-center prospective comparative study was carried out. The participants underwent a complex biochemical and hormonal examination, a hyperinsulinemic euglycemic and hyperglycemic clamps, a bioelectrical impedance analysis of the body composition before and 13 months after parathyroidectomy compared to sex-, age- and body mass index matched healthy volunteers. RESULTS: 45.8% of patients (n = 24) had excessive visceral fat. Insulin resistance was detected in 54.2% of cases. PHPT patients had higher serum triglycerides, lower M-value and higher C-peptide and insulin levels in both phases of insulin secretion compared to the control group (p < 0.05 for all parameters). There were tendencies to decreased fasting glucose (p = 0.031), uric acid (p = 0.044) and insulin levels of the second secretion phase (p = 0.039) after surgery, but no statistically significant changes of lipid profile and M-value as well as body composition were revealed. We obtained negative correlations between percent body fat and osteocalcin and magnesium levels in patients before surgery. CONCLUSION: PHPT is associated with insulin resistance that is the main risk factor of serious metabolic disorders. Surgery may potentially improve carbohydrate and purine metabolism.

Application of Neoaortoiliac System For Contained Rupture of Mycotic Infrarenal Aortic Aneurysm

Funding: None. Synopsis: 61-year-old male who initially presented to an outside facility with streptococcal pneumoniae meningitis and bacteremia. Of note, he had history of COVID-19 pneumonia a month prior. On hospital day 15, he reported sudden onset lower back pain prompting imaging which demonstrated a contained rupture of an infrarenal aortic aneurysm that had significantly evolved in comparison to admission imaging where his infrarenal aorta had the largest dimension measuring 2.9cm. We present the successful application of neoaortoiliac system (NAIS). Method(s): Proceeding with midline laparotomy we encountered dense adhesive disease due to his history of surgery for colon cancer. After adhesiolysis, we exposed the aorta and aneurysm with severe surrounding inflammatory changes. 20cm of femoral vein was harvested, reversed, and joined for a span of 4cm using an Endo GIA 45mm vascular load to create our neoaorta. Proximal and distal clamp zones were developed. Upon entering the aneurysm, a foul smell was encountered, revealing that the noxious process had destroyed the posterior wall of the aorta and paraspinal tissues. Our neoaorta was anastomosed in end-to-end fashion to the infrarenal aorta and subsequently to the common iliac arteries. Flow was initially restored to the hypogastric arteries and then the external iliac arteries. The retroperitoneum was closed over our repair and covered with omentum. Result(s): On post-operative day 2, he had hematochezia;intraoperatively, the IMA was noted to be 1mm in size, though had brisk back-bleeding and was ultimately ligated. A flexible sigmoidoscopy revealed ischemic sloughing of the sigmoid colon near his previous anastomosis from his colon cancer resection though no transmural necrosis. He remains on high-dose ceftriaxone to complete a 6-week course and metronidazole for 10 days due to his sigmoid mucosal ischemia per infectious disease recommendations. He is now post-operative day 10 and remains in the ICU. Conclusion(s): Mycotic aortic aneurysms constitute 1-1.8% of aortic aneurysms. The standard of treatment is aggressive debridement of involved aortic wall and periaortic tissue, in-situ or extra-anatomic reconstruction, coverage with an omental flap and long-term antibiotic therapy. NAIS is resistant to infection and aneurysmal dilation, however, is a time-consuming procedure with a mean completion time of 8 hours. Dorweiler et al. demonstrated that vascular reconstruction with femoral vein in infected aortoiliofemoral fields has a mortality of 9-10% with negligible rate of late complications (graft stenosis, thrombosis, and dilation) and that venous morbidity after femoral vein harvest is well tolerated. Clagett et al. demonstrated that NAIS fashioned from greater saphenous vein had a failure rate requiring intervention of 64% compared to 0% for those constructed with deep femoral vein. Lastly, it is important to note that our patient was previously COVID-19 positive. This case demonstrates that the sequela of COVID-19 may have been a significant factor in our patient's pathophysiology. As we continue to learn about the effects of COVID-19 on vascular pathology, we must keep a large repertoire of operative techniques at hand in order to treat complex presentations of vascular emergencies. [Formula presented] [Formula presented] [Formula presented] Institution: Orlando Health, Orlando, FLCopyright © 2022

COVID-19 Pandemic Adversely Affects the Provision of Desired Newborn Circumcision: Perinatal Physician Perspectives.

Over half of boys in the United States undergo circumcision, which has its greatest health benefits and lowest risks when performed during the newborn period under local anesthesia. The COVID-19 pandemic has affected delivery of patient care in many ways and likely also influenced the provision of newborn circumcisions. Prior to the pandemic, we planned to conduct a qualitative study to ascertain physician perspectives on providing newborn circumcision care. The interviews incidentally coincided with the onset of the pandemic and thus, pandemic-related changes emerged as a theme. We elected to analyze this theme in greater detail. Semi-structured interviews were conducted with perinatal physicians in a large urban city from 4/2020 to 7/2020. Physicians that perform or counsel regarding newborn circumcision and physicians with knowledge of or responsibility for hospital policies were eligible. Interviews were transcribed verbatim and qualitative coding was performed. Twenty-three physicians from 11 local hospitals participated. Despite no specific COVID-19 related questions in the interview guide, nearly half of physicians identified that the pandemic affected delivery of newborn circumcision care with 8 pandemic-related sub-themes. The commonest sub-themes included COVID-19 related changes in: (1) workflow processes, (2) staffing and availability of circumcision proceduralists, and (3) procedural settings. In summary, this qualitative study revealed unanticipated COVID-19 pandemic-related changes with primarily adverse effects on the provision of desired newborn circumcisions. Some of these changes may become permanent resulting in broad implications for policy makers that will likely need to adapt and redesign the processes and systems for the delivery of newborn circumcision care.

Local Anesthetic Like Inhibition of the Cardiac Na+ Channel Nav1.5 by Chloroquine and Hydroxychloroquine.

Introduction: Chloroquine (CQ) and its derivate hydroxychloroquine (HCQ) are successfully deployed for different diseases beyond the prophylaxis and treatment of malaria. Both substances exhibit antiviral properties and have been proposed for prophylaxis and treatment of COVID-19 caused by SARS-CoV-2. CQ and HCQ cause similar adverse events including life-threatening cardiac arrhythmia generally based on QT-prolongation, which is one of the most reported adverse events for both agents associated with the treatment of COVID-19. Various drugs known to induce QT-prolongation have been proven to exert local anesthetic (LA)-like properties regarding their impact on the cardiac Na+ channel Nav1.5. Inhibition of Nav1.5 is considered as the primary mechanism of cardiotoxicity caused by LAs. However, the mechanism of the arrhythmogenic effects of CQ and HCQ related to Nav1.5 has not yet been fully investigated. Therefore, the exact mechanism of how CQ and HCQ affect the sodium currents generated by Nav1.5 need to be further elucidated. Objective: This in vitro study aims to investigate the effects of CQ and HCQ on Nav1.5-generated sodium currents to identify possible LA-like mechanisms that might contribute to their arrhythmogenic properties. Methods: The effects of CQ and HCQ on Nav1.5-generated sodium currents by HEK-293 cells expressing either wild-type human Nav1.5 or mutant Nav1.5 F1760A are measured using the whole-cell patch-clamp technique. Results: Both agents induce a state-dependent inhibition of Nav1.5. Furthermore, CQ and HCQ produce a use-dependent block of Nav1.5 and a shift of fast and slow inactivation. Results of experiments investigating the effect on the LA-insensitive mutant Nav1.5-F1760A indicate that both agents at least in part employ the proposed LA-binding site of Nav1.5 to induce inhibition. Conclusion: This study demonstrated that CQ and HCQ exert LA-typical effects on Nav1.5 involving the proposed LA binding site, thus contributing to their arrhythmogenic properties.

Initiating a pediatric cardiac surgery program in europe during the COVID-19 pandemic: Methods, challenges and outcomes

Background and Aim: Before 2020, no pediatric cardiac surgery pro-gram was available at our institution, despite being a university hospital providing tertiary care for 6 million inhabitants. Our goal is to describe the preparation and the first year of expe-rience of our pediatric cardiac surgery program, which will even-tually cater for 300 patients annually. Method(s): The project was supported by European funds (INTERREG program). Medical and nursing staff training was performed via a transborder collaboration. Significant investments were necessary to reach the required standards for the facilities (operating rooms, pediatric intensive care beds) and equipment (cardiopulmonary bypass and ECMO machines, ultrasound sys-tems etc.). A multidisciplinary team was built over 3 years. The pediatric ECMO program was started a year prior to the surgical program. In parallel, a program dedicated to the study and care of neurological impact of congenital heart diseases and interventions was set up. Importantly, a progressive upscale was devised: only children with a weight gt;5 kg requiring non-complex surgeries were operated on during the first year. Result(s): The first year of experience was marked by challenges caused by the successive COVID-19 waves, such as restricted access to the operating room and a subsequent slow-down in the progression of the schedule. Fifty-nine patients constituted the cohort of the first year (October 2020-October 2021). In addition to low-risk procedures (left-to-right shunts closures etc.), cases included 6 tetralogy of Fallot repairs, 1 Ross procedure and 2 bilateral cavopulmonary connections. There were no early or late deaths. Median age was 6.3 years old (1.8-9.8) and median weight was 18.5 kg (10.0-32.0). Fourteen patients (23.7%) were operated on with a weight lt;10 kg. Bypass cases represented 72.9% (43 patients) of all cases. Median cardiopulmonary bypass and cross-clamping times were 88 (52-153) and 51 (26-98) minutes respectively. Median intensive care and hospital stays were 3 (2.0-6.7) and 6 days (5-11) respectively. Conclusion(s): Despite COVID-19-related difficulties, our pediatric cardiac surgery program achieved excellent outcomes in selected patients. Institutional support, meticulous planning, team cohesion and perseverance are keys for successful initiation of a program requiring such high technicality.

A NONINVASIVE RETRIEVAL METHOD FOR RETAINED GUIDEWIRE AFTER CENTRAL VENOUS LINE (CVL) PLACEMENT

SESSION TITLE: Imaging, ECMO, and other Procedures in the ICU Case Posters SESSION TYPE: Case Report Posters PRESENTED ON: 10/19/2022 12:45 pm - 01:45 pm INTRODUCTION: Central Venous Catheter placement is a common procedure in the ICU setting and provides a valuable route for medication delivery and venous access. The Seldinger method is the most commonly used method for placement of the catheter, and is the standard of care [1] with current recommendations. However, central line placement is still associated with complications including infection, thrombotic events, and mechanical malfunctions. Guidewire related complications are less common, but can occur [2]. Wire retention is a known, but avoidable complication of central venous catheter placement. Guidewire errors have been associated with operator fatigue, inexperience, and inadequate supervision of trainees. CASE PRESENTATION: An immunocompromised 40 year old female who presenting with hypoxia secondary to COVID-19, ultimately requiring intubation. She required initiation of continuous sedatives, analgesics, and vasopressors, for which a CVC was placed. The procedure proceeded in usual fashion with ultrasound and sterilization. Standard seldinger technique with US guidance was utilized. However, during advancement of the catheter, the wire was also advanced and lost within the catheter. DISCUSSION: Using ultrasound the wire could be seen within the lumen of the catheter and approximately 1-2 cm deep. A chest plain film was obtained and displayed above (Figure 1). Given the superficial location of the wire, bedside removal was attempted. Counter-traction was applied anterior to the catheter entry site with a second operator while suction was applied to the terminal catheter port using a 30 cc syringe. A debakey hemostat was utilized to clamp the catheter as it penetrated the dermis. The catheter was removed 2 cm and then the hemostat was released while still applying suction and then again replaced at the same site. This process was repeated three subsequent times and then the catheter was completely removed revealing the guidewire protruding from the initial entry site. The wire was safely removed. The patient was otherwise unharmed and would later discharge to rehab facility. CONCLUSIONS: Central venous catheter placement is a common ICU procedure than can be associated with complications. The above case reflects one complication that occurs per few thousands [3]. Fortunately, bedside retrieval was possible and further invasive procedures were avoided. The above method represents one possible method for removal of a guidewire that is only superficially buried. Finally, this case demonstrates that a thoughtful approach to procedural complications and use of available resources can avoid more invasive procedures, increased risk of further complications, and increased costs to the patient and healthcare system. Reference #1: Thaut L, Weymouth W, Hunsaker B, Reschke D. Evaluation of Central Venous Access with Accelerated Seldinger Technique Versus Modified Seldinger Technique. J Emerg Med. 2019 Jan;56(1):23-28. doi: 10.1016/j.jemermed.2018.10.021. Epub 2018 Nov 30. PMID: 30503723. Reference #2: Kornbau C, Lee KC, Hughes GD, Firstenberg MS. Central line complications. Int J Crit Illn Inj Sci. 2015;5(3):170-178. doi:10.4103/2229-5151.164940 Reference #3: Bessoud B, de Baere T, Kuoch V, Desruennes E, Cosset MF, Lassau N, Roche A. Experience at a single institution with endovascular treatment of mechanical complications caused by implanted central venous access devices in pediatric and adult patients. AJR Am J Roentgenol. 2003 Feb;180(2):527-32. doi: 10.2214/ajr.180.2.1800527. PMID: 12540466. DISCLOSURES: No relevant relationships by John Craver Scientific Medical Advisor relationship with Synspira Please note: 3 years Added 03/29/2022 by Bryan Garcia, value=Salary Speaker/Speaker's Bureau relationship with Insmed Please note: 3 years Added 03/29/2022 by Bryan Garcia, value=Honoraria Advisory Committee Member relationship with Zambon Pharmaceuticals Please note: 2 ears Added 03/29/2022 by Bryan Garcia, value=Honoraria No relevant relationships by John Murphy

Mitochondrial enzymes infused into the kidney under hydrodynamic pressures significantly improves creatinine and BUN levels at the onset of moderate and severe acute kidney injuries

Introduction: The COVID-19 pandemic has highlighted the need to address how renal insults are treated. There is an urgent need to better understand the complex relationship between infections and kidney disease and develop safe and effective approaches that can be translated to the clinic. Hydrodynamic fluid delivery has shown promise in influencing renal function in disease models. This technique previously provided preconditioned protection in acute injury models by upregulating the mitochondrial adaptation, while hydrodynamic injections of saline alone have also improved microvascular perfusion. Accordingly, hydrodynamic mitochondrial gene delivery was applied to investigate its ability to halt renal impairment that may occur following episodes of acute moderate and severe injuries in a rat model. Methods: Transgene infusates were prepared by suspending approximately 2 μg of IDH2 (isocitrate dehydrogenase 2 (NADP+) and mitochondrial) plasmid DNA/g of body weight in 0.5 ml of saline. Animals were subjected to moderate (bilateral pedicle clamp 30 mins) or severe (bilateral pedicle clamp 60 mins) forms of ischemia-reperfusion injury (IRI). Infusates were delivered directly into the left renal vein within 5 seconds, roughly 1 hour after IRI was established. Serum creatinine (SCr) and blood urea nitrogen (BUN) levels were monitored for 2 weeks. Results: Significant reductions in the levels of both metabolites (p < 0.05 for both cases) were achieved with single transgene treatments administered at both time points. Specifically, the maximal rises in SCr and BUN levels were reduced by at least 50%, which translated the effects of a severe injury to a moderate injury and a moderate injury to a mild injury. Conclusions: Therefore, this study identifies an approach that boosts recovery and halts the progression of ischemia-reperfusion at its inception and can be vital for high-risk conditions and may help devise translation models to address the rising incidence of acute renal diseases. No conflict of interest

A platform technology for generating subunit vaccines against diverse viral pathogens.

The COVID-19 pandemic response has shown how vaccine platform technologies can be used to rapidly and effectively counteract a novel emerging infectious disease. The speed of development for mRNA and vector-based vaccines outpaced those of subunit vaccines, however, subunit vaccines can offer advantages in terms of safety and stability. Here we describe a subunit vaccine platform technology, the molecular clamp, in application to four viruses from divergent taxonomic families: Middle Eastern respiratory syndrome coronavirus (MERS-CoV), Ebola virus (EBOV), Lassa virus (LASV) and Nipah virus (NiV). The clamp streamlines subunit antigen production by both stabilising the immunologically important prefusion epitopes of trimeric viral fusion proteins while enabling purification without target-specific reagents by acting as an affinity tag. Conformations for each viral antigen were confirmed by monoclonal antibody binding, size exclusion chromatography and electron microscopy. Notably, all four antigens tested remained stable over four weeks of incubation at 40°C. Of the four vaccines tested, a neutralising immune response was stimulated by clamp stabilised MERS-CoV spike, EBOV glycoprotein and NiV fusion protein. Only the clamp stabilised LASV glycoprotein precursor failed to elicit virus neutralising antibodies. MERS-CoV and EBOV vaccine candidates were both tested in animal models and found to provide protection against viral challenge.

Transient receptor potential melastatin 3 dysfunction in post COVID-19 condition and myalgic encephalomyelitis/chronic fatigue syndrome patients.

BACKGROUND: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a severe multisystemic condition associated with post-infectious onset, impaired natural killer (NK) cell cytotoxicity and impaired ion channel function, namely Transient Receptor Potential Melastatin 3 (TRPM3). Long-term effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has resulted in neurocognitive, immunological, gastrointestinal, and cardiovascular manifestations recently recognised as post coronavirus disease 2019 (COVID-19) condition. The symptomatology of ME/CFS overlaps significantly with post COVID-19; therefore, this research aimed to investigate TRPM3 ion channel function in post COVID-19 condition patients. METHODS: Whole-cell patch-clamp technique was used to measure TRPM3 ion channel activity in isolated NK cells of N = 5 ME/CFS patients, N = 5 post COVID-19 patients, and N = 5 healthy controls (HC). The TRPM3 agonist, pregnenolone sulfate (PregS) was used to activate TRPM3 function, while ononetin was used as a TRPM3 antagonist. RESULTS: As reported in previous research, PregS-induced TRPM3 currents were significantly reduced in ME/CFS patients compared with HC (p = 0.0048). PregS-induced TRPM3 amplitude was significantly reduced in post COVID-19 condition compared with HC (p = 0.0039). Importantly, no significant difference was reported in ME/CFS patients compared with post COVID-19 condition as PregS-induced TRPM3 currents of post COVID-19 condition patients were similar of ME/CFS patients currents (p > 0.9999). Isolated NK cells from post COVID-19 condition and ME/CFS patients were resistant to ononetin and differed significantly with HC (p < 0.0001). CONCLUSION: The results of this investigation suggest that post COVID-19 condition patients may have impaired TRPM3 ion channel function and provide further evidence regarding the similarities between post COVID-19 condition and ME/CFS. Impaired TRPM3 channel activity in post COVID-19 condition patients suggest impaired ion mobilisation which may consequently impede cell function resulting in chronic post-infectious symptoms. Further investigation into TRPM3 function may elucidate the pathomechanism, provide a diagnostic and therapeutic target for post COVID-19 condition patients and commonalities with ME/CFS patients.

Transatrial Balloon-Expandable Valve Implantation for Symptomatic Mitral Stenosis With Severe Annular Calcification—A Hybrid Solution

Background: Treatment of symptomatic mitral valve stenosis in severe mitral annular calcification is a surgical challenge. Transcatheter options include transfemoral transcatheter mitral valve replacement (TMVR), which poses its own risks, the most significant is left ventricular outflow tract (LVOT) obstruction. Transatrial hybrid TMVR optimizes advantages of both traditional open-heart surgery and transcatheter valve replacement. Methods: Retrospective review of seven high-risk patients (deemed ineligible for traditional surgery) undergoing transatrial implantation of a SAPIEN 3 valve (Edwards Lifesciences, Irvine, CA) in the mitral position for severe symptomatic mitral stenosis. Laceration of the Anterior Mitral leaflets to Prevent Outflow ObstructioN procedure was not considered due to heavy leaflet calcifications. Results: Seven patients treated consecutively from June 2020 to July 2021 were included in this analysis. Mean age was 77 years;six were females, one was male. Average STS score was 9.8. Three patients had New York Heart Association (NYHA) class IV heart failure. Mean left ventricular ejection fraction was 62%. Dominant mitral valve pathology included mitral stenosis in all patients. Mean mitral valve gradient was 12 mmHg. All patients had circumferential annular calcification except one, who had predominantly anterior calcification. All patients received the Edwards SAPIEN 3 valve and had anterior leaflet resection. Surgical approach was at the discretion of the attending cardiac surgeon. Mean cardiopulmonary bypass time was 85 minutes;mean cross-clamp time was 36 minutes. No anchoring felt was used. Technical success was 100%, with no device embolization. There was no clinically significant LVOT obstruction. There were two deaths: one occurred during index hospitalization due to worsening heart failure secondary to torrential tricuspid regurgitation, and the second was 2 months later due to COVID-19 infection. Conclusion: Surgical hybrid transatrial TMVR for patients at high surgical risk is technically feasible with high procedure success. A relatively shorter cardiac ischemic duration, direct visualization and resection of the anterior mitral leaflet can allow for safe TMVR without risk of LVOT obstruction.

Intubation Performance with Stylet and Preloaded Bougie for Rapid Sequence Intubation in Patients Undergoing General Anaesthesia: A Randomised Clinical Trial

Introduction: Rapid sequence induction requires quick and single attempt intubation to secure airway without any untoward complications. As the number of attempts increase, risk of desaturation and aspiration increase which is potentially life threatening. In such circumstances, miscalculation may cost loss of time which may prove fatal. Various adjuncts and techniques have been devised to prevent such calamities. Aim: To compare ease of intubation with angulated stylet versus distally preloaded bougie for rapid sequence intubation in elective general anaesthesia procedures. Materials and Methods: This randomised trial was conducted in 100 patients belonging to 18-60 years of age from November 2019 to October 2020. Patients were intubated using rapid sequence including cricoid pressure by either styletted endotracheal tube (Group S) or distally preloaded bougie (Group B), for surgeries performed under general anaesthesia. The primary outcome was to determine mean time to intubation (TTI) and number of attempts, while secondary outcomes were haemodynamic responses to intubation and complications. Data comparison between independent groups in this normally distributed data was done using student -t test while intragroup analysis was done using chi-square test. Results: A total of hundred patients were randomized into two groups- group S (mean age: 41.12 years) and group B (mean age: 37.34 years), of 50 patients each. Number of intubation attempts with stylet were single in 82%, two in 18% cases while with preloaded bougie, it was 80% and 14%, respectively (p-value=0.196). Time to intubation was 22.16 seconds (group S) versus 33.78 seconds (group B) (p-value <0.05). The haemodynamic assessments revealed that tachycardia, hypertension and increased End tidal carbon dioxide (EtCO2) was seen for 10 minutes immediately post induction in both the groups, though the intergroup difference was non significant. The incidence of sore throat was higher with stylet than bougie, though non-significant (p-value=0.118). Conclusion: Stylet should be preferred for ease of intubation in rapid sequence inductions. However, the insertion and removal of stylet must be done cautiously to prevent postoperative sore throat.

A Functional Interaction Between Y674-R685 Region of the SARS-CoV-2 Spike Protein and the Human α7 Nicotinic Receptor.

The α7 nicotinic acetylcholine receptor (nAChR) is present in neuronal and non-neuronal cells and has anti-inflammatory actions. Molecular dynamics simulations suggested that α7 nAChR interacts with a region of the SARS-CoV-2 spike protein (S), and a potential contribution of nAChRs to COVID-19 pathophysiology has been proposed. We applied whole-cell and single-channel recordings to determine whether a peptide corresponding to the Y674-R685 region of the S protein can directly affect α7 nAChR function. The S fragment exerts a dual effect on α7. It activates α7 nAChRs in the presence of positive allosteric modulators, in line with our previous molecular dynamics simulations showing favourable binding of this accessible region of the S protein to the nAChR agonist binding site. The S fragment also exerts a negative modulation of α7, which is evidenced by a profound concentration-dependent decrease in the durations of openings and activation episodes of potentiated channels and in the amplitude of macroscopic responses elicited by ACh. Our study identifies a potential functional interaction between α7 nAChR and a region of the S protein, thus providing molecular foundations for further exploring the involvement of nAChRs in COVID-19 pathophysiology.

Understanding Coronavirus-Related Delirium: Neurovirulent Cytokines and Their Impact on Neuronal Excitability

Rationale Delirium affects a majority of critically-ill patients, increasing mortality and dementia risk. The absence of effective therapy reversing neuronal changes in delirium emphasizes the need for greater understanding of delirium pathophysiology. Neuroinflammation represents a common pathway through which delirium-triggering diseases act. Glial cells sense systemic inflammation across the blood-brain barrier and become activated, releasing cytokines within the brain. In one disease model, systemic infection with neurovirulent coronaviruses caused delirium and specifically increased levels of TNF-α, IL-1β, IL-6, IL-12 and IL-15 in the mouse brain. Methods Here, we tested how neuronal function was affected in a coronavirus-induced neurovirulent cytokine (NVC) model of delirium. Using whole-cell patch clamp methods, we examined how single neuron excitability in murine primary neocortical cultures was impacted by 22-28 hour incubation in NVC. Results NVC treatment depolarized the resting membrane potential (RMP) compared to control (-65 ± 1.6 mV versus -73 ± 1 mV;P < 0.0001, n = 37 and 31 respectively) without affecting action potential characteristics. Delirium is often diagnosed due to altered responses to external stimuli. NVC exposure altered the sensitivity of neurons to changes in external Ca2+ and Mg2+ from physiological (1.1 mM, T1.1) to low (0.2 mM, T0.2) levels. The frequency of spontaneous firing was substantially increased following T0.2 application in control but not in NVC-treated neurons (p=0.026, ANOVA, control: 0.02 ± 0.01 Hz to 2.1 ± 1.2 Hz, n=16, p=0.046;NVC: 0.5 ± 0.4 Hz to 0.9 ± 0.3 Hz, n = 15, p=0.16). Consistent with this, evoked spiking following current injection was also observed in control but not NVC-treated neurons following the switch from T1.1 to T0.2 (P = 0.006, 2WRM ANOVA, Control: 3.9 ± 1.2 Hz vs. 8.5 ± 1.3 Hz n=37, p<0.0001;NVC: 4.8 ± 1.3 Hz vs. 4.5 ± 1.0 Hz, n=31, p=0.78). The attenuated excitability observed in NVC-treated neurons was reversed by hyperpolarization of the RMP. Evoked firing was substantially improved in NVC-treated cells after correcting the RMP (p =0.049, ANOVA, Control: 7.5 ± 1.8 Hz vs. 10 ± 2.5 Hz, n=15, p=0.25;NVC: 6.7 ± 2.2 Hz vs. 11.7 ± 2.8 Hz, n=16 p=0.01). Conclusion Our studies indicate that NVC-treated neurons have attenuated sensitivity to microenvironment changes. As these changes are reversible by correction of the RMP, further characterization of the underlying pathophysiological mechanism is essential to identify biologically plausible targets for delirium.

ECMO Specialist to Primer: Redefining the role of the RN and RT

Background: Since 2018, Methodist Hospital's ECMO program has rapidly expanded and now cares for almost 200 ECMO patients per year. In order to support this growth, an ECMO specialist pathway was created for RNs and RTs. At the inception of the ECMO specialist program, the training took an average of 6 months to complete. As the program expanded, and the COVID-19 pandemic ensued, we redefined the training and increased the frequency of courses offered to facilitate a large volume of specialists. The ECMO training has been effectively condensed and is now often completed in less than a month. In anticipation of further growth and the need for cost-efficient ECMO management, an ECMO primer role was created to operate in tandem with the ECMO team. This position redefines the role of the RN and RT, allowing them to offload perfusion services by assisting in initial cannulations, responding to ECMO related emergencies, overseeing the specialists, and building and priming circuits for future use. Methods: ECMO specialist: The specialists' role encompasses the 24-hour management of the circuit. This includes hourly circuit maintenance checks, accessing the circuit, moving an ECMO patient, and responding to circuit complications. The specialist is charged with knowing when to clamp, when to initiate the emergency back-up drive, when to emergently change the circuit, and how to respond to an acute decannulation. The training encompasses a 10-hour didactic training course and 2-day wet lab training. Didactic curriculum includes physiology, ECMO fundamentals, ECMO physiology, and ECMO management. 2-day wet lab training integrates live instruction with hands on training integrating the themes and concepts from the 10-hour course. The specialist candidates are asked to showcase their new skills on the second day in fully immersive, high-fidelity, case-based simulations. Upon completion, the specialist candidates are required to complete four shadow shifts with an ECMO specialist/primer. On the final shift, the specialist candidate will complete a skills validation before earning their ECMO specialist badge. ECMO primers: The primer role encompasses the oversight and management of all the ECMO patients and specialist, up to 24 patients. This includes collaborating in patient care and multidisciplinary rounds, management of equipment and rolling stock, educating and mentoring specialist, responding to emergencies, and advanced troubleshooting. The training encompasses baseline mastery of specialist skills, a 3-day wet lab, plus a minimum of 4 weeks of shadow shifts. The 3-day wet lab includes circuit building and priming, bedside initiation, cath lab conversions, advanced configurations, advanced troubleshooting, and circuit changes. Primers will utilize the highfidelity case-based simulations as well as cannulation simulations. Most of the primer training occurs at the bedside. Shadow shifts are scheduled with a chief primer and candidates are evaluated on bedside management, building and priming circuits, intra-hospital transport, ECMO initiation, circuit change, sterile back table set up, maximum barrier prep and drape, bedside cannulation, decannulation, and cannula reconfigurations. Candidates are formally evaluated biweekly and action plans are written to facilitate candidate success. Results: Since the launch of the program in July 2021, 280 specialists and 8 primers have completed training. By January 1st of 2022, the primer will fully offload the perfusionists in 24-hour coverage for non-CVOR ECMO management for up to 24 pumps at a time. Conclusion: By implementing both the ECMO specialist and ECMO primer roles, our ECMO program has continued to grow without compromising patient outcomes despite the pandemic. In addition to being more cost-efficient, creating an alternative growth pathway for RNs and RTs at the bedside has led to improved morale and increased staff retention.

Current Methods to Unravel the Functional Properties of Lysosomal Ion Channels and Transporters.

A distinct set of channels and transporters regulates the ion fluxes across the lysosomal membrane. Malfunctioning of these transport proteins and the resulting ionic imbalance is involved in various human diseases, such as lysosomal storage disorders, cancer, as well as metabolic and neurodegenerative diseases. As a consequence, these proteins have stimulated strong interest for their suitability as possible drug targets. A detailed functional characterization of many lysosomal channels and transporters is lacking, mainly due to technical difficulties in applying the standard patch-clamp technique to these small intracellular compartments. In this review, we focus on current methods used to unravel the functional properties of lysosomal ion channels and transporters, stressing their advantages and disadvantages and evaluating their fields of applicability.

The wandering bullet: An unusual case of paradoxical embolization involving the neck, aortic arch, and iliac artery

Paradoxical intravascular bullet embolism involving the aortic arch (AA) is a rare and highly lethal condition. We describe an unusual case of a civilian gunshot injury to the neck. A bullet entered in the neck, injured the internal jugular vein (IJV), and then continued into the lumen of the common carotid artery (CCA). The bullet traveled under its own momentum and against the flow of blood, along the carotid and brachiocephalic vessels, finally lodging in the wall of the lesser curvature of the AA. The injury tract resulted in an arterial-venous fistula between IJV and CCA and a pseudoaneurysm of the AA. Open surgical repair of the neck and AA was complicated by secondary distal embolization of the bullet, requiring an embolectomy.

Prolonged Exposure to Remdesivir Inhibits the Human Ether-a-go-go-related Gene Potassium Current

Introduction: Remdesivir is indicated for the treatment of COVID-19 in patients requiring hospitalization. However, cases of QTc interval prolongation and torsade de pointes (TdP) have been reported to the FDA Adverse Event Reporting System. Drug-induced QTc prolongation and TdP is the single most common cause of withdrawal, relabeling and use restriction of marketed drugs. Most drugs that prolong the QTc inhibit a potassium current (IKr), which is encoded by the human ether-a-go-go-related gene (hERG) and is crucial for ventricular repolarization and action potential duration. Research Question or Hypothesis: To assess the potential for remdesivir and its metabolite, GS441524, to inhibit hERG-related currents. Study Design: Cell-based hERG Assay Methods: Whole-cell, voltage-clamp experiments were performed in HEK-293 cells stably expressing hERG. Borosilicate glass electrodes (resistance: 2-4 MW) filled with internal solution were used to record tail currents at depolarizing and repolarizing voltages tail current. To assess acute effects, drugs were added to the internal pipette solution, and for prolonged exposure;cells were incubated with remdesivir for 24 hours prior to recording. Results: Acute exposure to remdesivir and GS-441524 did not significantly inhibit peak activation or maximum tail current density. However, prolonged exposure to remdesivir 100 nM and 1 mM, but not 10 nM, inhibited peak activation currents by 32% (19±2 pA/pF, p = 0.03) and 36% (18±2 pA/pF, p = 0.02) respectively. Remdesivir 100 nM and 1 mM, also inhibited the maximum tail current density by 40% (18±2 pA/pF, p = 0.02) and 37% (19±2 pA/pF, p = 0.03), respectively. Conclusion: Prolonged exposure to physiological concentrations of remdesivir inhibits hERG-related currents. These results, coupled with clinical reports of QTc prolongation and TdP, highlight the need for a rigorous assessment of the effect of remdesivir on ventricular repolarization and risk of proarrhythmia.

In vitro ion channel profile and ex vivo cardiac electrophysiology properties of the R(-) and S(+) enantiomers of hydroxychloroquine.

Hydroxychloroquine (HCQ) is a derivative of the antimalaria drug chloroquine primarily prescribed for autoimmune diseases. Recent attempts to repurpose HCQ in the treatment of corona virus disease 2019 has raised concerns because of its propensity to prolong the QT-segment on the electrocardiogram, an effect associated with increased pro-arrhythmic risk. Since chirality can affect drug pharmacological properties, we have evaluated the functional effects of the R(-) and S(+) enantiomers of HCQ on six ion channels contributing to the cardiac action potential and on electrophysiological parameters of isolated Purkinje fibers. We found that R(-)HCQ and S(+)HCQ block human Kir2.1 and hERG potassium channels in the 1 µM-100 µM range with a 2-4 fold enantiomeric separation. NaV1.5 sodium currents and CaV1.2 calcium currents, as well as KV4.3 and KV7.1 potassium currents remained unaffected at up to 90 µM. In rabbit Purkinje fibers, R(-)HCQ prominently depolarized the membrane resting potential, inducing autogenic activity at 10 µM and 30 µM, while S(+)HCQ primarily increased the action potential duration, inducing occasional early afterdepolarization at these concentrations. These data suggest that both enantiomers of HCQ can alter cardiac tissue electrophysiology at concentrations above their plasmatic levels at therapeutic doses, and that chirality does not substantially influence their arrhythmogenic potential in vitro.