The role of high cholesterol in SARS-CoV-2 infectivity.

Coronavirus disease 2019 (COVID-19) is a respiratory infection caused by severe acute respiratory syndrome coronavirus 2. The virus binds to angiotensinogen converting enzyme 2 (ACE2), which mediates viral entry into mammalian cells. COVID-19 is notably severe in the elderly and in those with underlying chronic conditions. The cause of selective severity is not well understood. Here we show cholesterol and the signaling lipid phosphatidyl-inositol 4,5 bisphosphate (PIP2) regulate viral infectivity through the localization of ACE2's into nanoscopic (<200 nm) lipid clusters. Uptake of cholesterol into cell membranes (a condition common to chronic disease) causes ACE2 to move from PIP2 lipids to endocytic ganglioside (GM1) lipids, where the virus is optimally located for viral entry. In mice, age and high-fat diet increase lung tissue cholesterol by up to 40%. And in smokers with chronic disease, cholesterol is elevated 2-fold, a magnitude of change that dramatically increases infectivity of virus in cell culture. We conclude increasing the ACE2 location near endocytic lipids increases viral infectivity and may help explain the selective severity of COVID-19 in aged and diseased populations.

Hydroxychloroquine blocks SARS-CoV-2 entry into the endocytic pathway in mammalian cell culture.

Hydroxychloroquine (HCQ), a drug used to treat lupus and malaria, was proposed as a treatment for SARS-coronavirus-2 (SARS-CoV-2) infection, albeit with controversy. In vitro, HCQ effectively inhibits viral entry, but its use in the clinic has been hampered by conflicting results. A better understanding of HCQ's mechanism of actions in vitro is needed. Recently, anesthetics were shown to disrupt ordered clusters of monosialotetrahexosylganglioside1 (GM1) lipid. These same lipid clusters recruit the SARS-CoV-2 surface receptor angiotensin converting enzyme 2 (ACE2) to endocytic lipids, away from phosphatidylinositol 4,5 bisphosphate (PIP2) clusters. Here we employed super-resolution imaging of cultured mammalian cells (VeroE6, A549, H1793, and HEK293T) to show HCQ directly perturbs clustering of ACE2 receptor with both endocytic lipids and PIP2 clusters. In elevated (high) cholesterol, HCQ moves ACE2 nanoscopic distances away from endocytic lipids. In cells with resting (low) cholesterol, ACE2 primarily associates with PIP2 clusters, and HCQ moves ACE2 away from PIP2 clusters-erythromycin has a similar effect. We conclude HCQ inhibits viral entry through two distinct mechanisms in high and low tissue cholesterol and does so prior to inhibiting cathepsin-L. HCQ clinical trials and animal studies will need to account for tissue cholesterol levels when evaluating dosing and efficacy.

The role of high cholesterol in age-related COVID19 lethality.

Coronavirus disease 2019 (COVID19) is a respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) originating in Wuhan, China in 2019. The disease is notably severe in elderly and those with underlying chronic conditions. A molecular mechanism that explains why the elderly are vulnerable and why children are resistant is largely unknown. Here we show loading cells with cholesterol from blood serum using the cholesterol transport protein apolipoprotein E (apoE) enhances the entry of pseudotyped SARS-CoV-2 and the infectivity of the virion. Super resolution imaging of the SARS-CoV-2 entry point with high cholesterol shows almost twice the total number of endocytic entry points. Cholesterol concomitantly traffics angiotensinogen converting enzyme (ACE2) to the endocytic entry site where SARS-CoV-2 presumably docks to efficiently exploit entry into the cell. Furthermore, in cells producing virus, cholesterol optimally positions furin for priming SARS-CoV-2, producing a more infectious virion with improved binding to the ACE2 receptor. In vivo, age and high fat diet induces cholesterol loading by up to 40% and trafficking of ACE2 to endocytic entry sites in lung tissue from mice. We propose a component of COVID19 severity based on tissue cholesterol level and the sensitivity of ACE2 and furin to cholesterol. Molecules that reduce cholesterol or disrupt ACE2 localization with viral entry points or furin localization in the producer cells, may reduce the severity of COVID19 in obese patients.

Association between Tunneled Pleural Catheter Use and Infection in Patients Immunosuppressed from Antineoplastic Therapy. A Multicenter Study.

Rationale: Patients with malignant or paramalignant pleural effusions (MPEs or PMPEs) may have tunneled pleural catheter (TPC) management withheld because of infection concerns from immunosuppression associated with antineoplastic therapy.Objectives: To determine the rate of infections related to TPC use and to determine the relationship to antineoplastic therapy, immune system competency, and overall survival (OS).Methods: We performed an international, multiinstitutional study of patients with MPEs or PMPEs undergoing TPC management from 2008 to 2016. Patients were stratified by whether or not they underwent antineoplastic therapy and/or whether or not they were immunocompromised. Cumulative incidence functions and multivariable competing risk regression analyses were performed to identify independent predictors of TPC-related infection. Kaplan-Meier method and multivariable Cox proportional hazards modeling were performed to examine for independent effects on OS.Results: A total of 1,408 TPCs were placed in 1,318 patients. Patients had a high frequency of overlap between antineoplastic therapy and an immunocompromised state (75-83%). No difference in the overall (6-7%), deep pleural (3-5%), or superficial (3-4%) TPC-related infection rates between subsets of patients stratified by antineoplastic therapy or immune status was observed. The median time to infection was 41 (interquartile range, 19-87) days after TPC insertion. Multivariable competing risk analyses demonstrated that longer TPC duration was associated with a higher risk of TPC-related infection (subdistribution hazard ratio, 1.03; 95% confidence interval [CI], 1.00-1.06; P = 0.028). Cox proportional hazards analysis showed antineoplastic therapy was associated with better OS (hazard ratio, 0.84; 95% CI, 0.73-0.97; P = 0.015).Conclusions: The risk of TPC-related infection does not appear to be increased by antineoplastic therapy use or an immunocompromised state. The overall rates of infection are low and comparable with those of immunocompetent patients with no relevant antineoplastic therapy. These results support TPC palliation for MPE or PMPEs regardless of plans for antineoplastic therapy.

Preliminary clinical experience with Calypso anchored beacons for tumor tracking in lung SBRT.

PURPOSE: To present our preliminary experience with the recently released Calypso lung beacons to track lung tumor location during stereotactic body radiation therapy (SBRT). MATERIALS/METHODS: Five recent lung SBRT patients had Calypso lung beacons implanted for tumor tracking during treatment. Beacons were placed by a pulmonologist using fluoroscopic navigation within 1 week prior to planning four-dimensional computed tomography (4DCT) acquisition. Patients were immobilized in a full-body double-vacuum bag. For the first three patients, a verification 4DCT was obtained prior to the first fraction with the patient in the treatment position to assess both beacon migration and motion of tumor and beacons relative to planning day. For each treatment fraction, Calypso was used to position the patient. A verification cone-beam CT (CBCT) confirmed the Calypso-defined target position was appropriate. Real-time Calypso tracking information was also acquired and compared to an action level that was used to determine if the tumor migrated outside of the planning target volume. RESULTS: For four patients, the implant procedure was well tolerated, with average CBCT-based shifts being within 0.2 mm of the shifts reported by Calypso at the time of imaging. The other patient had a small pneumothorax due to very peripheral tumor location and experienced beacon migration. However, the patient quickly recovered from the pneumothorax, and after deactivating that beacon, motion tracking was possible throughout his treatment. CONCLUSIONS: All patients were successfully treated with SBRT using the newly released Calypso lung beacons, with initial positioning confirmed by this clinic's current clinical standard of CBCT. The system allowed us to validate, with real-time confirmation, that the planned internal target volumes were appropriate to each day's extent of actual tumor motion. An efficient and effective workflow for utilizing the new lung beacons for SBRT treatments was developed.

Core needle biopsy with endobronchial ultrasonography: single center experience with 100 cases.

INTRODUCTION: Adequate sampling by endobronchial ultrasound (EBUS)-transbronchial needle aspiration to meet the demands of precision medicine or histologic evaluation is challenging. There is increasing demand for core biopsy specimens with advances in therapy. Franseen enodoscopic ultrasound needles have shown promising results in gastroenterology application for obtaining core biopsies and same design has recently been extended for pulmonary use. We evaluated Franseen needles with EBUS to assess its utility, safety and ability to provide core biopsy specimens. MATERIALS AND METHODS: Retrospective analysis of our database at the University of Utah of patients undergoing EBUS with a Franseen needle was performed to ascertain the performance characteristics of this needle in the first 100 patients after its implementation. Medical records were also reviewed to identify any immediate procedure-related complications. RESULTS: One hundred seventy locations were sampled in 100 patients. A total of 152 lymph nodes and 18 masses were sampled. Core biopsies, as per pathology report, were seen in 87% of patients. A clinically concordant pathological diagnosis was established in 97% of patients. Diagnostic yield for granulomatous lymphadenopathy was 95.6% (22 of 23). No patient-related adverse events were noted. CONCLUSION: The Franseen needle evaluated in this study can safely procure core tissue samples during EBUS bronchoscopy that are adequate for histopathological diagnosis in benign and malignant lesions. Its ability to provide adequate tissue in patients with granulomatous inflammation is encouraging.

Cholesterol and COVID19 lethality in elderly.

Coronavirus disease 2019 (COVID19) is a respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) originating in Wuhan, China in 2019. The disease is notably severe in elderly and those with underlying chronic conditions. A molecular mechanism that explains why the elderly are vulnerable and why children are resistant is largely unknown. Here we show loading cells with cholesterol from blood serum using the cholesterol transport protein apolipoprotein E (apoE) enhances the entry of pseudotyped SARS-CoV-2 and the infectivity of the virion. Super resolution imaging of the SARS-CoV-2 entry point with high cholesterol shows almost twice the total number of endocytic entry points. Cholesterol concomitantly traffics angiotensinogen converting enzyme (ACE2) to the endocytic entry site where SARS-CoV-2 presumably docks to efficiently exploit entry into the cell. Furthermore, in cells producing virus, cholesterol optimally positions furin for priming SARS-CoV-2, producing a more infectious virion with improved binding to the ACE2 receptor. In vivo, age and high fat diet induces cholesterol loading by up to 40% and trafficking of ACE2 to endocytic entry sites in lung tissue from mice. We propose a component of COVID19 severity based on tissue cholesterol level and the sensitivity of ACE2 and furin to cholesterol. Molecules that reduce cholesterol or disrupt ACE2 localization with viral entry points or furin localization in the producer cells, may reduce the severity of COVID19 in obese patients.

An Age-Dependent Sequence of Physiological Processes Defines Developmental Root Senescence.

Aging-related processes in plant tissues are associated with changes in developmental and physiological processes relevant for stress tolerance and plant performance. While senescence-regulated processes have been extensively characterized in leaves, they remain poorly described in roots. Here, we investigated the physiological processes and molecular determinants underlying the senescence of seminal roots in hydroponically grown barley (Hordeum vulgare). Transcriptome profiling in apical and basal root tissues revealed that several NAC-, WRKY-, and APETALA2 (AP2)-type transcription factors were upregulated just before the arrest of root elongation, when root cortical cell lysis and nitrate uptake, as well as cytokinin concentrations ceased. At this time point, root abscisic acid levels peaked, suggesting that abscisic acid is involved in root aging-related processes characterized by expression changes of genes involved in oxidative stress responses. This temporal sequence of aging-related processes in roots is highly reminiscent of typical organ senescence, with the exception of evidence for the retranslocation of nutrients from roots. Supported by the identification of senescence-related transcription factors, some of which are not expressed in leaves, our study indicates that roots undergo an intrinsic genetically determined senescence program, predominantly influenced by plant age.

Indwelling Pleural Catheter versus Pleurodesis for Malignant Pleural Effusions. A Systematic Review and Meta-Analysis.

RATIONALE: Several randomized trials have compared the efficacy of an indwelling pleural catheter (IPC) versus the more traditional chemical pleurodesis in the management of malignant pleural effusion (MPE). OBJECTIVES: As part of the American Thoracic Society's guidelines for management of MPE, we performed a systematic review and a meta-analysis to compare patient-centered outcomes with the use of a tunneled pleural catheter versus chemical pleurodesis for the first-line treatment of malignant pleural effusions. METHODS: We performed literature searches in MEDLINE, Embase, and the Cochrane Central Register of Controlled Trials. We included randomized controlled trials comparing IPC and pleurodesis in adult patients with symptomatic MPE. Risk of bias was assessed with the Cochrane Risk of Bias tool recommended by the Cochrane Methods Bias Group. The meta-analysis was performed with Review Manager software, using a random effects model. We used risk ratios (RRs) with 95% confidence interval (CI) as the effect measure for dichotomous outcomes and mean differences for continuous outcomes. RESULTS: We identified five randomized trials, involving 545 patients, that compared IPC and pleurodesis. Lack of blinding and the inevitable attrition of patients due to death resulted in an overall high risk of bias among the studies. No differences in survival or measures of dyspnea were observed in any of the studies. Total hospital length of stay was shorter, and repeat pleural interventions were less common in the IPC group (RR, 0.32; 95% CI, 0.18-0.55). However, the risk of cellulitis was higher with IPC (RR, 5.83; 95% CI, 1.56-21.8). No differences were noted in other adverse events. CONCLUSIONS: Compared with chemical pleurodesis, IPC results in shorter hospital length of stay and fewer repeat pleural procedures but carries a higher risk of cellulitis. Careful assessment of individual patient preferences and costs should be considered when choosing between IPC and pleurodesis.

Management of Malignant Pleural Effusions. An Official ATS/STS/STR Clinical Practice Guideline.

BACKGROUND: This Guideline, a collaborative effort from the American Thoracic Society, Society of Thoracic Surgeons, and Society of Thoracic Radiology, aims to provide evidence-based recommendations to guide contemporary management of patients with a malignant pleural effusion (MPE). METHODS: A multidisciplinary panel developed seven questions using the PICO (Population, Intervention, Comparator, and Outcomes) format. The GRADE (Grading of Recommendations, Assessment, Development and Evaluation) approach and the Evidence to Decision framework was applied to each question. Recommendations were formulated, discussed, and approved by the entire panel. RESULTS: The panel made weak recommendations in favor of: 1) using ultrasound to guide pleural interventions; 2) not performing pleural interventions in asymptomatic patients with MPE; 3) using either an indwelling pleural catheter (IPC) or chemical pleurodesis in symptomatic patients with MPE and suspected expandable lung; 4) performing large-volume thoracentesis to assess symptomatic response and lung expansion; 5) using either talc poudrage or talc slurry for chemical pleurodesis; 6) using IPC instead of chemical pleurodesis in patients with nonexpandable lung or failed pleurodesis; and 7) treating IPC-associated infections with antibiotics and not removing the catheter. CONCLUSIONS: These recommendations, based on the best available evidence, can guide management of patients with MPE and improve patient outcomes.

Publisher Correction: Combating subclonal evolution of resistant cancer phenotypes.

The originally published version of this Article contained an error in Figure 4. In panel a, grey boxes surrounding the subclones associated with patients #2 and #4 obscured adjacent portions of the heatmap. This error has now been corrected in both the PDF and HTML versions of the Article.

Primary Pulmonary Carcinoid Tumor: A Long-term Single Institution Experience.

OBJECTIVES: Primary carcinoid tumors of the lung are rare tumors which comprise approximately 0.5% to 5% of all lung malignancies in adults and roughly 20% to 30% of all carcinoid tumors. The purpose of this retrospective, descriptive study was to describe the incidence, characteristics, and outcomes of patients treated for primary pulmonary carcinoid tumor at a single institution. MATERIALS AND METHODS: All patients with a diagnosis of primary pulmonary carcinoid tumor treated from 1989 to 2009 were reviewed. Data collected included demographics, pathology, tobacco use, clinical presentation, tumor location, tumor spread, treatment, and survival. RESULTS: There were 59 cases of pulmonary carcinoid tumors: 47 typical (80%) and 12 atypical (20%). All but 4 patients underwent surgery, including 54 (92%) lung-sparing resections and 1 pneumonectomy. Five of 55 patients received concurrent adjuvant chemoradiation therapy; 4 patients with atypical and 1 with typical histology. Three additional patients with atypical carcinoid were treated only with adjuvant radiotherapy, palliative radiotherapy, or palliative chemotherapy, respectively. The Kaplan-Meier 5- and 10-year overall survivals were both 80% within the entire population. In the 88% of patients who achieved complete remission, disease-free survival was 98%. A review of a large series from the literature is also presented. CONCLUSIONS: Surgical resection was primary and adequate therapy for most typical carcinoid tumors with high overall survival and disease-free survival. Adjuvant chemotherapy or radiotherapy might be considered for patients with atypical carcinoid tumors who present with adverse pathologic findings.

NR5A1 mutations are not associated with male infertility in Indian men.

NR5A1 or steroidogenic factor 1 (SF1) is an autosomal gene, which encodes a protein that is a member of nuclear receptor family. NR5A1 regulates the transcription of numerous genes that are expressed in hypothalamic-pituitary-gonadal axis and adrenal cortex which in turn, coordinate the gonadal development, steroidogenesis and sex differentiation. Several mutations in NR5A1 have been reported to cause gonadal dysgenesis with adrenal insufficiency in individuals with 46,XY karyotype. However, studies in the past few years have shown that NR5A1 mutations can also contribute to primary ovarian insufficiency and impaired spermatogenesis. As there is no genetic study on NR5A1 in Indian infertile men, we have sequenced the entire coding region (exons 2-7) of NR5A1 in 502 infertile men of which, 414 were non-obstructive azoospermic and 88 severe oligozoospermic, along with 427 ethnically matched fertile controls. Interestingly, none of the mutations reported to be associated with male infertility were found in our study, except one polymorphism, rs1110061. However, it was not significantly different between infertile and fertile groups (p = .76). In addition, we have identified six intronic variants; but none of them was significantly associated with male infertility.

Combating subclonal evolution of resistant cancer phenotypes.

Metastatic breast cancer remains challenging to treat, and most patients ultimately progress on therapy. This acquired drug resistance is largely due to drug-refractory sub-populations (subclones) within heterogeneous tumors. Here, we track the genetic and phenotypic subclonal evolution of four breast cancers through years of treatment to better understand how breast cancers become drug-resistant. Recurrently appearing post-chemotherapy mutations are rare. However, bulk and single-cell RNA sequencing reveal acquisition of malignant phenotypes after treatment, including enhanced mesenchymal and growth factor signaling, which may promote drug resistance, and decreased antigen presentation and TNF-α signaling, which may enable immune system avoidance. Some of these phenotypes pre-exist in pre-treatment subclones that become dominant after chemotherapy, indicating selection for resistance phenotypes. Post-chemotherapy cancer cells are effectively treated with drugs targeting acquired phenotypes. These findings highlight cancer's ability to evolve phenotypically and suggest a phenotype-targeted treatment strategy that adapts to cancer as it evolves.

Endodontic Management of a Maxillary First Molar with Seven Root Canal Systems Evaluated Using Cone-Beam Computed Tomography Scanning.

Permanent maxillary first molar morphology has been studied widely. Usually, it has three roots with three canals and the frequent deviation being incidence of a second mesiobuccal canal, thus showing a multifaceted root canal anatomy. Other variations comprise four and five roots and abnormal root canal morphology within individual roots. Many case reports with five and six root canals or a C-shaped canal configuration have been reported in the past. This varying number of root canals poses a challenge for endodontist in detecting and treating by root canal treatment. This problem can be avoided using techniques such as using the dental operating microscope and cone-beam computed tomography (CBCT). We report a change in canal morphology case of maxillary first molar having seven root canals, its evaluation, and management using latest equipment such as CBCT.