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Recent clinical trials have investigated the use of fluvoxamine in preventing clinical deterioration in nonhospitalized patients with acute COVID-19 infection via stimulation of sigma-1 receptors, which regulates cytokine production and functional inhibition of acid sphingomyelinase activity, which may prevent infection of epithelial cells with SARS-CoV-2. However, the role of fluvoxamine is currently unclear because of a paucity of studies, particularly because the drug is being repurposed as an immunomodulatory and antiviral agent.Aim of our meta-analysis was to investigate the efficacy of fluvoxamine in nonhospitalized patients with acute COVID-19 infection.Comprehensive literature search of PubMed, Embase, Cochrane Library databases, and Web of Science was performed from inception to February 10, 2022, for studies comparing fluvoxamine versus placebo for outpatient management of COVID-19.The primary outcome of interest was rate of hospitalization. The secondary outcomes were rates of patients requiring mechanical ventilation and mortality. The random-effects model was used to calculate the risk ratios (RR) and confidence intervals (CI). A P value0.05 was considered statistically significant. Heterogeneity was assessed using the Higgins I2 index.Three studies (2 randomized controlled trials and one prospective cohort trial) involving 1762 patients were included in the meta-analysis. In patients who received fluvoxamine compared with placebo, there was no significant difference in rates of hospitalization (RR 0.26, 95% CI, 0.04-1.73, P = 0.16, I2 = 62%), mechanical ventilation (RR 0.73, 95% CI, 0.45-1.19, P = 0.21, I2 = 0%), and mortality (RR 0.67, 95% CI, 0.37-1.22, P = 0.19, I2 = 0%).Current evidence does not indicate a significant effect of fluvoxamine on the rates of hospitalization, mechanical ventilation, and mortality of patients with COVID-19 infection.

Supplementary Table 1. Linkage disequilibrium patterns of the tag-SNPs used for genotyping the 6q23.3 locus. Supplementary Table 2. Cytogenetic and molecular information on AML patients used for functional studies (n=12). Supplementary Table 3. French-American-British (FAB) classification35 and molecular information on AML patients used to determine miR-3662's abundance (n=8). Supplementary Table 4. Canonical pathway analysis of the miR-3662-associated gene expression signature. Supplementary Figure 1. Transcription factor binding according to the transcription factor chip data from ENCODE. Supplementary Figure 2. Electrophoretic mobility shift assay comparing the binding affinity of the alleles of rs66650371 and rs9483788. Supplementary Figure 3. Endogenous miR-3662 expression levels of hematopoietic progenitor (HP) cells during differentiation, total bone marrow aspirate of three non-leukemic donors (total BM 1-3), different populations of differentiated peripheral blood cells, and three AML cell lines. Supplementary Figure 4. Top panel, Macroscopic pictures of the spleens of three mice of the scramble and miR-3662-infected groups (organs harvested post-mortem). All mice had a massive splenomegaly compared to the un-injected, sacrificed control mouse. Bottom panel, images of spleen histologies (40x enlargement). Slides were stained for CD45 to proof MV4-11 origin of the leukemia. Supplementary Figure 5. Endogenous abundance of miR-3662 and IKBKB in patient samples and cell lines. Supplementary Figure 6. Comparison of the relative miR-3662 abundance of AML patient blasts and AML cell lines before (black) and after (red) forced miR-3662 expression with the lentiviral expression construct.

Observational studies and randomized controlled trials (RCTs) have shown conflicting outcomes for multiple arterial graft (MAG) coronary artery bypass graft surgery compared with single arterial grafts (SAGs). The predominant evidence supporting the use of MAGs is observational. The aim of this meta-analysis of RCTs is to compare outcomes following MAG and SAG. We searched multiple databases for RCTs comparing MAG versus SAG. The clinical outcomes studied were all-cause mortality, cardiac mortality, myocardial infarction (MI), revascularization, stroke, sternal wound complications, and major bleeding. We used hazard ratio (HR), relative risk (RR), and corresponding 95% confidence interval (CI) for measuring outcomes. Ten RCTs (6392 patients) were included. The average follow-up in the studies was 4.2 years. The average age of the patients in the studies ranged from 56.3 years to 74.6. No significant difference was seen between MAG and SAG groups for all-cause mortality (11.8% vs 12.7%, HR 0.94, 95% CI 0.81 to 1.09, p 0.36), cardiac mortality (4.1% vs 4.5%, HR 0.96 95% CI 0.74 to 1.26, p 0.77), MI (3.5% vs 5.1%, HR 0.87 95% CI 0.67 to 1.12, p 0.28), and major bleeding (3.3% vs 4.9%, RR 0.85 95% CI 0.64 to 1.13, p 0.26). Repeat revascularization in MAG showed a lower RR than SAG when one of the confounding studies was excluded (RR 0.63, 95% CI 0.4 to 0.99, p 0.04). The incidence of stroke was lower in MAG than SAG (2.9% vs 3.9%, RR 0.74 95% CI 0.56 to 0.98, p 0.03). MAG had higher incidence of sternal wound complications than SAG (2.9% vs 1.7%, RR 1.75 95% CI 1.19 to 2.55, p 0.004). In conclusion, MAG does not have a survival advantage compared with SAG but is better in revascularization and risk of stroke. This benefit may be set off by a higher incidence of sternal wound complications in MAG.

In the COVID-19 pandemic, to minimize aerosol-generating procedures, cardiac magnetic resonance imaging (CMR) was utilized at our institution as an alternative to transesophageal echocardiography (TEE) for diagnosing infective endocarditis (IE). This retrospective study evaluated the clinical utility of CMR for detecting IE among 14 patients growing typical microorganisms on blood cultures or meeting modified Duke Criteria. Seven cases were treated for IE. In 2 cases, CMR results were notable for possible leaflet vegetations and were clinically meaningful in guiding antibiotic therapy, obtaining further imaging, and/or pursuing surgical intervention. In 2 cases, vegetations were missed on CMR but detected on TEE. In 3 cases, CMR was non-diagnostic, but patients were treated empirically. There was no difference in antibiotic duration or outcomes over 1 year. CMR demonstrated mixed results in diagnosing valvular vegetations and guiding clinical decision-making. Further prospective controlled trials of CMR Vs TEE are warranted.

The effects of cardiovascular comorbidities on outcomes in COVID-19 hospitalized patients has not been well studied.This is a hospital-based study evaluating the effects of CVD on the outcomes in patients admitted with COVID-19. Clinical outcomes were studied in patients with and without CVD.Eighty-seven patients had CVD, and 193 patients had no history of CVD. Ischemic heart disease was the most common CVD (63%). When compared with patients with no CVD, those with CVD had higher mortality (29% vs 9%, p 0.001), discharge to a skilled nursing facility (SNF) (36% vs 15%, p 0.001), and change of code status to 'do not resuscitate' (41% vs 14%, p 0.001). The odds for mortality were high with ischemic heart disease (OR 3.6, 95% CI 1.8-7.3, p 0.001), and systolic heart failure (OR 3.8,95% CI 1.2-12.3, p = 0.02). Patients in the CVD group were more likely to have incident atrial fibrillation (22% vs 3%, p 0.001), type 2 Mi (17% vs 6%, p = 0.002), high BNP (57% vs 14%, p 0.001), acute kidney injury (64% vs 29%, p 0.001), and any type of circulatory shock (27% vs 12%, p = 0.001).CVD is associated with increased mortality, myocardial injury, arrhythmias, and discharges to an SNF.

Introduction Atrial fibrillation is a common cardiac arrhythmia that affects approximately 2% of the overall population. Guidelines suggest the use of anti-arrhythmic agents as initial therapy in patients with symptomatic atrial fibrillation, however using cryoablation as a first line therapy might have increased efficacy. The safety and efficacy of cryoablation as initial therapy has not yet been established. Purpose We performed a systematic review and meta-analysis of randomized controlled trials to investigate the use of cryoballoon catheter ablation compared to anti-arrhythmic therapy as an initial intervention to prevent recurrence of atrial tachyarrhythmias in patients with atrial fibrillation. We also wanted to determine if using this initial ablative approach did not present increased adverse events. Methods A comprehensive search of multiple databases was performed to find randomized control trials that directly compared cryoablation therapy versus anti-arrhythmic therapy as initial treatment for patients with atrial fibrillation. A total of three RCTs met the inclusion criteria (724 patients) and were used in the meta-analysis. The primary outcome of our meta-analysis was recurrence of atrial tachyarrhythmias. The secondary outcome evaluated serious adverse events of each therapy. Results The results showed a statistically significant reduction of recurrence of atrial tachyarrhythmic events in patients receiving cryoablation compared to anti-arrhythmic therapy [Risk Ratio (RR): 0.60, 95% CI (0.49, 0.72), P Conclusion Our meta-analysis showed that cryoablation therapy as an initial therapy is more efficacious than anti-arrhythmic therapy in patients with atrial fibrillation without an increased risk of serious adverse events. Funding Acknowledgement Type of funding sources: None.

Background There is limited data on the clinical characteristics and predictors of mortality of coronavirus disease-2019 (COVID-19) in North West Ohio. We performed a retrospective review of patients hospitalized with COVID-19 in the ProMedica Health System in Northwest Ohio from March 25 to June 16, 2020. The study aims to identify epidemiological, clinical characteristics, and predictors of Mortality of COVID-19 patients in Northwest Ohio. Methods This study was conducted on 217 COVID-19 patients admitted to ProMedica Health System Hospitals in Northwest Ohio from March 25 to June 16, 2020. We collected data, including clinical signs, symptoms, and outcomes of the COVID-19 patients. We compared clinical signs and symptoms along with comorbidities of survivors and non-survivors. Results Of the 217 patients included in the study, the mean age of the population was 63.13 (SD 17.8), of which 194 (89.4%, mean age 61.7 years) survived while 23 (10.6%, mean age 74.6 years) died. Among them, 53% were females and 47% male. Common presenting symptoms were chest pain (91.71%), shortness of breath (79.7%), cough (71%), and fever (64%). Mortality was associated with age greater than 63 (p-value 0.0052) and hypertension (p-value: 0.0058) with marginal significance with gender (p-value: 0.0642), chest pain (p-value: 0.0944), and history of cancer (p-value: 0.0944). Conclusions Advanced age and hypertension (HTN) are independent predictors for increased mortality. History of cancer and chest pain are associated with increased mortality with marginal significance. Awareness among physicians about predictors of mortality is essential in dealing with COVID-19 patients. It is essential to educate the public about preventative strategies such as wearing masks to decrease mortality and morbidity from this pandemic.

Vascular calcification (VC) is one of the major causes of cardiovascular morbidity and mortality in patients with chronic kidney disease (CKD). VC is a complex process expressing similarity to bone metabolism in onset and progression. VC in CKD is promoted by various factors not limited to hyperphosphatemia, Ca/Pi imbalance, uremic toxins, chronic inflammation, oxidative stress, and activation of multiple signaling pathways in different cell types, including vascular smooth muscle cells (VSMCs), macrophages, and endothelial cells. In the current review, we provide an in-depth analysis of the various kinds of VC, the clinical significance and available therapies, significant contributions from multiple cell types, and the associated cellular and molecular mechanisms for the VC process in the setting of CKD. Thus, we seek to highlight the key factors and cell types driving the pathology of VC in CKD in order to assist in the identification of preventative, diagnostic, and therapeutic strategies for patients burdened with this disease.

Infective endocarditis (IE) carries a high mortality rate. Consequently, the prognosis is poorer in patients with multiple valve involvement. Due to poor prognosis of patients with endocarditis, early diagnosis and management of these patients can be challenging in the clinical setting. We describe a case of a 45-year-old man who came in with bacteremia secondary to a diabetic foot ulcer. Electrocardiogram (EKG) showed complete third-degree heart block which rose suspicion for possible valvular abscess formation. Transthoracic echocardiogram (TTE) was performed and revealed vegetations on the aortic and mitral valve. A follow-up transesophageal echocardiogram (TEE) showed an abscess on the aortic valve along with vegetations on the mitral and tricuspid valve, the latter which was missed on TTE. The prompt utilization of TEE in detecting early and late mechanical complications of endocarditis is imperative in facilitating rapid clinical decision-making and early intervention. Patients with multi-valve endocarditis are at extremely high risk of complications and should be evaluated for surgical intervention immediately.

The coronavirus disease 2019 (COVID-19) pandemic continues to cause significant morbidity and mortality worldwide. This study aims to identify specific lab markers, complications, and treatments that may be associated with increased mortality in COVID-19 patients. This study is retrospective in nature; it included 217 COVID-19 positive patients who were admitted to a ProMedica Health System hospital in Northwest Ohio, United States, between March 25 and June 16, 2020. We collected various laboratory values, complications, and treatment courses. T test and χ2 analyses were used to predict mortality. COVID-19 test was confirmed via polymerase chain reaction. Of 217 patients included in the study, the mean age of the population was 63.13 (SD, 17.8), of which 194 (89.4%, mean age 61.7 years) survived while 23 (10.6%, mean age 74.6 years) died. Among them, 53% were females and 47% male. Laboratory values that were associated with mortality were low hemoglobin (p = .0046), elevated INR (p = .0005), low platelets (p = .0246) and elevated procalcitonin (p = .0472). Marginally significant laboratory values included elevated troponin (p = .0661), and elevated creatinine (p = .0741). Treatment with either antibiotic, antifungals, antivirals, blood transfusion, steroids, and intubation were all statistically significant for mortality. COVID-19 related complications with either ARDS, myocarditis, elevated INR, septic shock, or age greater than 63 were significant predictors of mortality. Low hemoglobin, elevated INR, Low platelet, elevated procalcitonin, treated with either antibiotic, antifungal, antiviral, blood transfusion, steroids, and intubation are associated with high mortality related to COVID-19 infection. Healthcare professionals must be aware of these predictors.

Introduction The cause-and-effect relationship of QTc prolongation in Coronavirus disease 2019 (COVID-19) patients has not been studied well. Objective We attempt to better understand the relationship of QTc prolongation in COVID-19 patients in this study. Methods This is a retrospective, hospital-based, observational study. All patients with normal baseline QTc interval who were hospitalized with the diagnosis of COVID-19 infection at two hospitals in Ohio, USA were included in this study. Results Sixty-nine patients had QTc prolongation, and 210 patients continued to have normal QTc during hospitalization. The baseline QTc intervals were comparable in the two groups. Patients with QTc prolongation were older (mean age 67 vs. 60, P 0.003), more likely to have underlying cardiovascular disease (48% versus 26%, P 0.001), ischemic heart disease (29% versus 17%, P 0.026), congestive heart failure with preserved ejection fraction (16% versus 8%, P 0.042), chronic kidney disease (23% versus 10%, P 0.005), and end-stage renal disease (12% versus 1%, P Conclusion Older age, ESRD, underlying cardiovascular disease, potential virus mediated cardiac injury, and drugs like hydroxychloroquine/azithromycin, contribute to QTc prolongation in COVID-19 patients. The role of ACEi in preventing QTc prolongation in COVID-19 patients needs to be studied further.

Introduction Cirrhosis is known to be an important prognostic factor in determining morbidity and mortality in preoperative cardiac risk assessment for cardiac surgery. Data is limited on outcomes in patients with infective endocarditis (IE) and comorbid liver cirrhosis. The objective of our study is to evaluate the clinical outcomes in patients suffering from IE both with and without underlying liver cirrhosis as well as to determine rates of in-hospital mortality and factors that contribute to this outcome. Hypothesis Liver cirrhosis worsens clinical outcomes in patients with IE. Materials and methods Patients with a principal diagnosis of IE with and without liver cirrhosis were identified by querying the Healthcare Cost and Utilization (HCUP) database, specifically the National Inpatient Sample for the years 2013 and 2014 using International Classification of Diseases, Ninth Revision (ICD-9) codes. Results During 2013 and 2014, a total of 17,952 patients were admitted with a diagnosis of IE, out of whom 780 had concurrent liver cirrhosis. There was increased in-hospital mortality [15.6% vs 10.2%, aOR = 1.57 (1.27-1.93)], acute kidney injury [41.4% vs 32.6%, aOR = 1.45 (1.24-1.69)], and hematologic complications [32.1 vs 14.7%, aOR = 2.87 (2.44-3.37)] in patients with IE with liver cirrhosis when compared to patients with IE without liver cirrhosis. Patients having IE without liver cirrhosis underwent an increased number of interventions, i.e. aortic (7.2 vs 3.7%, aOR = 0.51 (0.34-0.76)) and mitral (4.9% vs 3.4%, aOR = 0.39 (0.23-0.69)) valvular replacements as compared to those with liver cirrhosis, which may explain the increased mortality seen in patients with liver cirrhosis. Conclusion Liver cirrhosis is an important prognostic risk factor for in-hospital mortality in patients with IE. The coagulopathic state in addition to increased rates of bleeding complications and renal dysfunction make these patients poor surgical candidates thus contributing to higher mortality. Further research into the individual risk factors contributing to the increased mortality rates in patients with IE and cirrhosis is required.

Minor conduction abnormalities such as first-degree heart blocks are generally overlooked on electrocardiogram (EKG) as their impact on clinical management is usually not substantial. However, they can be an important screening tool for early diagnosis of infective endocarditis (IE) and associated perivalvular complications, especially in patients with surgical valve replacements. This case report describes a 58-year-old male with a past medical history of bicuspid aortic valve status post replacement five years prior to presentation who initially presented with presumed symptoms of a complicated urinary tract infection (UTI) and later developed chest pain and shortness of breath. He showed no initial signs of infection including negative blood and urine cultures. EKG showed new onset prolonged PR interval. He then underwent a transthoracic echocardiogram (TTE) which showed prosthetic valve dysfunction and subsequently underwent transesophageal echocardiogram (TEE) which revealed vegetations on all leaflets and circumferential peri-aortic abscess encompassing both coronary ostia and extending towards the tricuspid and mitral valve leaflets. The patient then underwent redo-sternotomy for dissection of mediastinal adhesions, extraction of the aortic bio-prosthesis, and debridement of the aortic root abscess. The aortic root was replaced with a homograft and the valve cultures were positive for Enterococcus faecium. The patient developed complete heart block afterwards and received a permanent pacemaker; repeat cultures showed no further evidence of infection. This case report is presented to reiterate the importance of early detection of IE-related aortic valve abscess and their rare sequelae. Early screening for conduction abnormalities via EKG and subsequently a TEE can allow prompt identification and management of valvular abnormalities to prevent life-threatening complications and improve patient outcomes.

Objective: Cardiotonic steroids (CTS) are known ligands of the Na+/K+-ATPase (NKA) and chronic elevations in volume expanded conditions such as hypertension and chronic kidney disease (CKD) can mediate a pro-inflammatory and pro-fibrotic phenotype. Paraoxonase1 (PON1) is a lactonase enzyme that circulates bound to HDL and possesses antioxidant, anti-inflammatory and anti-atherogenic properties. PON1 can hydrolyze CTS to inactive open-ring forms making them incapable of stimulating NKA and initiating pro-inflammatory signaling cascades. Diminished PON1 activity results in detrimental clinical outcomes in the context of CKD but whether reduced PON1 activity is mechanistically linked to adverse cardiovascular events in CKD remains unknown. We hypothesized that PON-1 can attenuate progression of cardiac inflammation in CKD via modulating the pathogenic pathways induced by CTS signaling using a well characterized Dahl salt-sensitive rat model of hypertensive renal disease and elevated CTS. Methods: Dahl salt-sensitive wild type ( SS-WT ), PON1 knockout ( SS-PON1KO ), and PON1 knockout rats treated with 3E9 anti-CTS antibody (3E9 mAb,50μg/kg i.p. every week for a total of 4 injections) were fed a high salt (8%) diet for five weeks to induce hypertensive renal disease and elevate CTS levels. H&E staining was performed on hearts to analyze immune cell infiltration. Real-time PCR (RT-PCR) analysis was performed for markers of inflammation (IL6, IL1β, and CCL2), hypertrophy (Myh7, NPPA, and Slc8a), and fibrosis (Timp1). Results: RT-PCR analysis revealed significantly increased (≥1.5 fold) expression of cardiac inflammatory (pSS-PON1 KO compared to SS-WT rats after high salt feeding. Treatment of SS-PON1 KO rats with 3E9 mAb significantly decreased (≥ 2.5 fold) expression of Timp1, IL6, Ccl2, IL1β, NPPA, Myh7 and Slc8a (all pSS-PON1KO rats treated with 3E9 mAb compared to SS-PON-1 KO rats (3.5±0.5 VS1.813±0.4094,p Conclusion: Our findings suggest that PON-1 via its counter-regulatory mechanism of the CTS signaling axis exhibits a cardioprotective role in chronic kidney disease.

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disorder that affects multiple systems throughout the body. Although there are multiple documented vasculopathies that can be seen in NF1, there are very few documented cases of coronary artery aneurysms with complete thrombosis of the ectatic vessel resulting in myocardial infarction. This case report describes a 28-year-old male with a past medical history of NF1 who presented with an anterolateral ST-segment elevation myocardial infarction. He underwent urgent cardiac catheterization, which was significant for severe thrombotic occlusion of the mid-left anterior descending artery (LAD) with thrombolysis in myocardial infarction (TIMI) flow 0. The LAD was noted to be severely ectatic. Percutaneous coronary intervention (PCI) with thrombectomy was attempted and was unsuccessful, with TIMI flow 0 after the intervention attempt. An echocardiogram was performed, which showed left ventricular ejection fraction (LVEF) of 30%-35%. This case report is presented to familiarize physicians with the rare vasculopathies that can occur in patients with NF1. Occlusive or aneurysmal disease can occur almost anywhere in the body in patients with NF1 due to the proliferation of fusiform endothelial cells in the blood vessels.

Coronavirus disease 2019 (COVID-19) caused infection in 168,000 cases worldwide in about 148 countries and killed more than 6,610 people around the world as of March 16, 2020, as per the World Health Organization (WHO). Compared to severe acute respiratory syndrome and Middle East respiratory syndrome, there is the rapid transmission, long incubation period, and disease containment is becoming extremely difficult. The main aim of this systematic review is to provide a comprehensive clinical summary of all the available data from high-quality research articles relevant to the epidemiology, demographics, trends in hospitalization and outcomes, clinical signs and symptoms, diagnostic methods and treatment methods of COVID-19, thus increasing awareness in health care providers. We also discussed various preventive measures to combat COVID-19 effectively. A systematic and protocol-driven approach is needed to contain this disease, which was declared as a global pandemic on March 11, 2020, by the WHO.

Delayed coking is one of the most widely used residue upgradation process in crude oil refining where vacuum residue is thermally cracked and converted into distillates and petroleum coke. During heating of vacuum residue in coker furnace, coke is continuously deposited in furnace tubes which is removed at regular intervals by one of the three methods namely (i) pigging, (ii) steam air decoking or (iii) online spalling (OLS). OLS is one of the preferred methods of coke removal from coker furnace tubes. We have noticed very effective as well as “not so effective” OLS in a commercial delayed coker furnace. In present article, the changes in composition and properties of furnace coke during effective and ineffective OLS were systematically analyzed by various analytical tools such as Dilatometer, TGA, HRSEM, SEM-EDX and XRD. Based on these analyses, it was found that inorganic deposits such as iron sulphides and tube metal corrosion products play a major role in failure of OLS. Higher total acid number (TAN) of vacuum residue may be promoting the formation of iron sulfide deposits in furnace tube.

Chromosomal aberrations and multiple genome-wide association studies (GWAS) have established a major hematopoietic quantitative trait locus in chromosome 6q23.3. The locus comprises an active enhancer region, in which some of the associated SNPs alter transcription factor binding. We now identify miR-3662 as a new functional driver contributing to the associated phenotypes. The GWAS SNPs are strongly associated with higher miR-3662 expression. Genome editing of rs66650371, a three-base-pair deletion, suggests a functional link between the SNP genotype and the abundance of miR-3662. Increasing miR-3662′s abundance increases colony formation in hematopoietic progenitor cells, particularly the erythroid lineage. In contrast, miR-3662 is not expressed in acute myeloid leukemia cells, and its overexpression has potent antileukemic effects in vitro and in vivo. Mechanistically, miR-3662 directly targets NF-κB–mediated transcription. Thus, miR-3662 is a new player of the hematopoietic 6q23.3 locus. Significance: The characterization of miR-3662 has identified a new actor in the prominent hematopoietic quantitative trait locus in chromosome 6q23.3. The mechanistic insights into miR-3662′s function may reveal novel or only partially known pathways for normal and malignant hematopoietic cell proliferation. Cancer Discov; 6(9); 1036–51. ©2016 AACR. This article is highlighted in the In This Issue feature, p. 932

It was recently discovered that the NRAS isoform 5 (20 amino acids) is expressed in melanoma and results in a more aggressive cell phenotype. This novel isoform is responsible for increased phosphorylation of downstream targets such as AKT, MEK, and ERK as well as increased cellular proliferation. This structure report describes the NMR solution structure of NRAS isoform 5 to be used as a starting point to understand its biophysical interactions. The isoform is highly flexible in aqueous solution, but forms a helix-turn-coil structure in the presence of trifluoroethanol as determined by NMR and CD spectroscopy.

It was recently discovered that the NRAS isoform 5 (20 amino acids) is expressed in melanoma and results in a more aggressive cell phenotype. This novel isoform is responsible for increased phosphorylation of downstream targets such as AKT, MEK, and ERK as well as increased cellular proliferation. This structure report describes the NMR solution structure of NRAS isoform 5 to be used as a starting point to understand its biophysical interactions. The isoform is highly flexible in aqueous solution, but forms a helix‐turn‐coil structure in the presence of trifluoroethanol as determined by NMR and CD spectroscopy.

The protein NRAS is part of the NRAS-BRAF-MEK-ERK signaling cascade. Recently, 5 NRAS isoforms were discovered by our group, and it was observed that isoform 5 overexpression in vitro resulted in a more aggressive cell phenotype. NRAS isoform 5 increases phosphorylation of downstream targets (AKT, MEK, and ERK). The structural report is unavailable for NRAS isoform 5. In addition, it was found in the current study that the NRAS isoform 5 does not have GTPase activity due to lack the canonical GTP binding region. Given the importance of this pathway in driving melanoma progression, we studied the structural details of this novel NRAS isoform 5 by two biophysical techniques, NMR and CD spectroscopy. The circular dichroism spectra of NRAS isoform 5 that was C-acetylated and N-amidated was measured using 11%, 14%, 21%, 45%, 56% and 85% trifluoroethanol (TFE) in a phosphate buffer. The secondary structural elements were induced at ∼14-15% TFE. There were no qualitative differences in the CD spectra between 56% and 85% TFE. Therefore 56% deuterated TFE was utilized in the NMR structural studies. Two dimensional (2D) homonuclear and heteronuclear experiments were acquired at 25°C with Bruker Avance III HD 600 and 800 MHz NMR spectrometers (Campus Chemical Instrument Center NMR facility), each equipped with a triple resonance z axis gradient TXI cryoprobe for amino acid chemical shift assignments and structural determination of NRAS isoform 5. The NMR chemical shifts for the protons were assigned using traditional 2D TOCSY, COSY, and NOESY experiments. The 2D proton-carbon HSQC was utilized to facilitate the assignment of the alpha and side chain aliphatic protons. In the absence of TFE, there were no observable NOEs that could represent ordered secondary structures. Other than intra residue and short range sequential NOEs (i-j ≤ 2), only a weak medium range NOE was observed from the aromatic protons of Tyr 4 to the gamma proton of Val 7/8. The secondary structure of NRAS isoform 5 was determined via the same NMR techniques in 56% TFE. The secondary structure was defined by 69 sequential NOEs and 39 medium range NOEs. NOEs characteristic of helical structure were observed in the NOESY spectra. For example, NOEs were measured from H(alpha, i) to H(beta, i+3) for residues Tyr 4, Lys 5, and Leu 6. The tertiary fold was determined by 10 key long-range NOEs. The flexible C terminal is brought in close proximity to the N-terminal helix by 4 NOEs between Val 8/9 and Val 14, and 5 NOEs between Tyr 4 and Try 20 to form a helix-turn-coil structure in presence of TFE. In addition, the proton chemical shifts for the aromatic proton chemical shifts for the tyrosine aromatic ring became distinct. NRAS isoform 5 is highly flexible in aqueous solution, but forms a helix-turn-coil structure in the presence of trifluoroethanol as determined by NMR and CD spectroscopy. This data may be utilized as a starting point to understand the biophysical interactions of this novel NRAS isoform. Citation Format: Joseph Markowitz, Tapas K. Mal, Chunhua Yuan, Nicholas B. Courtney, Mitra Patel, Andrew R. Stiff, James Blachly, Christopher Walker, Ann-Kathrin Eisfeld, Albert de la Chapelle, William E. Carson. 3D structural report of NRAS isoform 5. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3093.