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Abstract: Background: Next-generation sequencing (NGS) of circulating tumor DNA (ctDNA) has emerged as a powerful complement to tissue NGS, offering a noninvasive and serially conductible test. Its application holds promise in enhancing the assessment of spatial and temporal molecular tumor heterogeneity, thus providing valuable insights into cancer progression and treatment response. In this study, we explore the mutational landscape of renal cell carcinoma (RCC) patients through comprehensive profiling of mutations in ctDNA and matched tissue samples, aiming to elucidate the concordance and clinical significance of molecular alterations detected in both circulating and tissue-derived DNA. Methods: From the Tempus multimodal database, we retrospectively analyzed de-identified NGS data from patients with RCC that had dual tissue (Tempus xT, 648 genes) and ctDNA testing (Tempus xF, 105 genes). Patients with matched samples (collected +/- 90 days of one another) were included. We evaluated socio-demographic and clinical characteristics and select pathogenic somatic short variants (PSSV) and copy number variants [(amplifications and deletions, two copy number losses (CNL)]. Concordance analyses were restricted to the 105 genes tested on the ctDNA panel and further restricted to short variants, with the exception of amplifications and CNL detected by both xF and xT. Analysis was further stratified by metastatic status (n=260, metastatic, n=120 non-metastatic) prior to collection of both xT and xF. Results: Among all patients (n=393), the median age was 61 years, and 71% were male. The patient cohort comprised a diverse population based on race, with 75% white, 12% African American, 4.8% Asian and 8% others. The median time from tissue to blood collection was 21 days (IQR, 7, 39). 67% (n=265) and 68% (n=266) had metastatic disease at the time of tissue and blood collection, respectively. The most common tissue sites were kidney (49%, n=189), bone (11%, n=43), lung (9%, n=34), lymph node (8%, n=29), liver (6%, n=23), and brain/CNS (4%, n=17). Genes harboring the most common PSSV in tissue included VHL (59% n=232), PBRM1 (31%, n=123), SETD2 (23%, n=91), TP53 (14%, n=54), BAP1 (12%, n=46) and TERT (11%, n=45). Genes with common PSSV in ctDNA included TP53 (23%, n=91), VHL (18%, n=69), BAP1 (6%, n=23), PBRM1 (5%, n=21), PTEN (4%, n=15), KRAS (4%, n=14) and NF2 (4%, n=14). The combination of tissue and ctDNA testing increased the detection of mutations (Table 1). There was higher concordance between somatic alterations in select genes among patients with metastases vs. non-metastases, including BAP1 (55.9% vs. 9.1%), TP53 (36.8% vs. 9.1%), VHL (32.3% vs. 12.5%), ARID1A (25% vs. 16.7%), and ATM (25% vs. 0%). Table 1: Detection of genetic mutations in tissue and ctDNA testing Conclusions: The analysis conducted in this study highlights the complementary nature of ctDNA profiling alongside tissue-based NGS in RCC, demonstrating an increased detection of mutations. Particularly, we observed a higher concordance between ctDNA and tissue profiling in individuals with metastatic disease, suggesting the potential utility of ctDNA analysis in advanced stages of RCC. Further research is warranted to elucidate how longitudinal ctDNA analysis can delineate biomarkers of response and resistance at both the mutation and ctDNA fraction levels. Understanding these dynamics could offer valuable insights into disease progression and guide personalized treatment strategies for RCC patients. DOD CDMRP Funding: no

BackgroundLittle is known whether electronic cigarettes (ECIG) increase vulnerability to future atherosclerotic cardiovascular disease. We determined, using an ex vivo mechanistic atherogenesis assay, whether proatherogenic changes including monocyte transendothelial migration and monocyte-derived foam cell formation are increased in people who use ECIGs.MethodsIn a cross-sectional single-center study using plasma and peripheral blood mononuclear cells from healthy participants who are nonsmokers or with exclusive use of ECIGs or tobacco cigarettes (TCIGs), autologous peripheral blood mononuclear cells with patient plasma and pooled peripheral blood mononuclear cells from healthy nonsmokers with patient plasma were utilized to dissect patient-specific ex vivo proatherogenic circulating factors present in plasma and cellular factors present in monocytes. Our main outcomes were monocyte transendothelial migration (% of blood monocyte cells that undergo transendothelial migration through a collagen gel) and monocyte-derived foam cell formation as determined by flow cytometry and the median fluorescence intensity of the lipid-staining fluorochrome BODIPY in monocytes of participants in the setting of an ex vivo model of atherogenesis.ResultsStudy participants (N=60) had median age of 24.0 years (interquartile range [IQR], 22.0-25.0 years), and 31 were females. Monocyte transendothelial migration was increased in people who exclusively used TCIGs (n=18; median [IQR], 2.30 [ 1.29-2.82]; P

The impact of tobacco cigarette (TCIG) smoking and electronic cigarette (ECIG) vaping on the risk of development of severe COVID-19 is controversial. The present study investigated levels of proteins important for SARS-CoV-2 pathogenesis present in plasma because of ectodomain shedding in smokers, ECIG vapers, and non-smokers (NSs). Protein levels of soluble angiotensin-converting enzyme 2 (ACE2), angiotensin (Ang) II (the ligand of ACE2), Ang 1-7 (the main peptide generated from Ang II by ACE2 activity), furin (a protease that increases the affinity of the SARS-CoV-2 spike protein for ACE2), and products of ADAM17 shedding activity that predict morbidity in COVID-19 (IL-6/IL-6R alpha (IL-6/IL-6Rα) complex, soluble CD163 (sCD163), L-selectin) were determined in plasma from 45 NSs, 30 ECIG vapers, and 29 TCIG smokers using ELISA. Baseline characteristics of study participants did not differ among groups. TCIG smokers had increased sCD163, L-selectin compared to NSs and ECIG vapers (p  0.1 for all comparisons). Further studies are needed to determine if increased furin activity and ADAM17 shedding activity that is associated with increased plasma levels of sCD163 and L-selectin in healthy young TCIG smokers may contribute to the future development of severe COVID-19 and cardiovascular complications of post-acute COVID-19 syndrome.

PurposeThe underlying premise of prostate cancer active surveillance (AS) is that cancers likely to metastasize will be recognized and eliminated before cancer-related disease can ensue. Our study was designed to determine the prostate cancer upgrading rate when biopsy guided by magnetic resonance imaging (MRGBx) is used before entry and during AS.Materials and methodsThe cohort included 519 men with low- or intermediate-risk prostate cancer who enrolled in prospective studies (NCT00949819 and NCT00102544) between February 2008 and February 2020. Subjects were preliminarily diagnosed with Gleason Grade Group (GG) 1 cancer; AS began when subsequent MRGBx confirmed GG1 or GG2. Participants underwent confirmatory MRGBx (targeted and systematic) followed by surveillance MRGBx approximately every 12 to 24 months. The primary outcome was tumor upgrading to ≥GG3.ResultsUpgrading to ≥GG3 was found in 92 men after a median followup of 4.8 years (IQR 3.1-6.5) after confirmatory MRGBx. Upgrade-free probability after 5 years was 0.85 (95% CI 0.81-0.88). Cancer detected in a magnetic resonance imaging lesion at confirmatory MRGBx increased risk of subsequent upgrading during AS (HR 2.8; 95% CI 1.3-6.0), as did presence of GG2 (HR 2.9; 95% CI 1.1-8.2) In men who upgraded ≥GG3 during AS, upgrading was detected by targeted cores only in 27%, systematic cores only in 25% and both in 47%. In 63 men undergoing prostatectomy, upgrading from MRGBx was found in only 5 (8%).ConclusionsWhen AS begins and follows with MRGBx (targeted and systematic), upgrading rate (≥GG3) is greater when tumor is initially present within a magnetic resonance imaging lesion or when pathology is GG2 than when these features are absent.

IntroductionThe severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the virus responsible for the COVID-19 pandemic, gains entry into the host cell when its Spike protein is cleaved by host proteases TMPRSS2 and furin, thereby markedly increasing viral affinity for its receptor, angiotensin-converting enzyme-2 (ACE2). In rodent and diseased human lungs, tobacco cigarette (TCIG) smoke increases ACE2, but the effect of electronic cigarette vaping (ECIG) is unknown. It is unknown whether nicotine (in both TCIGs and ECIGs) or non-nicotine constituents unique to TCIG smoke increase expression of key proteins in COVID-19 pathogenesis.MethodsImmune (CD45+) cells collected before the pandemic in otherwise healthy young people, including TCIG smokers (n = 9), ECIG vapers (n = 12), or nonsmokers (NS) (n = 12), were studied. Using flow cytometry, expression of key proteins in COVID-19 pathogenesis were compared among these groups.ResultsTCIG smokers and ECIG vapers had similar smoking or vaping burdens as indicated by similar plasma cotinine levels. TCIG smokers compared with NS had a significantly increased percentage of cells that were positive for ACE2 (10-fold, p < .001), TMPRSS2 (5-fold, p < .001), and ADAM17 (2.5-fold, p < .001). Additionally, the mean fluorescence intensity (MFI) consistently showed greater mean ACE2 (2.2-fold, p < .001), TMPRSS2 (1.5-fold, p < .001), furin (1.1-fold, p < .05), and ADAM17 (2-fold, p < .001) in TCIG smokers compared with NS. In ECIG vapers, furin MFI was increased (1.15-fold, p < .05) and TMPRSS2 MFI tended to be increased (1.1-fold, p = .077) compared with NS.ConclusionsThe finding that key instigators of COVID-19 infection are lower in ECIG vapers compared with TCIG smokers is intriguing and warrants additional investigation to determine if switching to ECIGs is an effective harm reduction strategy. However, the trend toward increased proteases in ECIG vapers remains concerning.Implications(1) This is the first human study to report a marked increase in proteins critical for COVID-19 infection, including ACE2, TMPRSS2, and ADAM17, in immune cells from healthy tobacco cigarette smokers without lung disease compared with e-cigarette vapers and nonsmokers. (2) These findings warrant additional investigation to determine whether switching to electronic cigarettes may be an effective harm reduction strategy in smokers addicted to nicotine who are unable or unwilling to quit. (3) The increase in proteases in electronic cigarette vapers remains concerning.

This randomized clinical crossover trial evaluates the association of a single session of electronic cigarette vaping with cellular oxidative stress in healthy young people who do not smoke compared with individuals with long-term tobacco cigarette or electronic cigarette use.

[Figure: see text].

It is unclear how oxidative stress triggered by smoking and vaping may alter specific immune cell subsets. In this study, we showed that tobacco cigarette smoking, but not electronic-cigarette vaping, is associated with increased expression of major proteins in the toll-like receptor 4 (TLR4) inflammasome-interleukin (IL)-6 signalling axis in monocyte subtypes and T cells. TLR4 senses oxidative stress in immune cells caspase-1 is a key protein of inflammasome activation, and IL-6R-α is the receptor for IL-6 that drives proatherogenic IL-6 signalling. These findings implicate the non-nicotine, pro-oxidant toxicants in tobacco cigarette smoke as instigators of increased expression of key proteins in the TLR4-inflammasome-IL-6 axis that contribute to atherogenesis.

Background Tobacco cigarettes (TCs) increase oxidative stress and inflammation, both instigators of atherosclerotic cardiac disease. It is unknown if electronic cigarettes (ECs) also increase immune cell oxidative stress. We hypothesized an ordered, "dose-response" relationship, with tobacco-product type as "dose" (lowest in nonsmokers, intermediate in EC vapers, and highest in TC smokers), and the "response" being cellular oxidative stress (COS) in immune cell subtypes, in otherwise, healthy young people. Methods and Results Using flow cytometry and fluorescent probes, COS was determined in immune cell subtypes in 33 otherwise healthy young people: nonsmokers (n=12), EC vapers (n=12), and TC smokers (n=9). Study groups had similar baseline characteristics, including age, sex, race, and education level. A dose-response increase in proinflammatory monocytes and lymphocytes, and their COS content among the 3 study groups was found: lowest in nonsmokers, intermediate in EC vapers, and highest in TC smokers. These findings were most striking in CD14dimCD16+ and CD14++CD16+ proinflammatory monocytes and were reproduced with 2 independent fluorescent probes of COS. Conclusions These findings portend the development of premature cardiovascular disease in otherwise healthy young people who chronically vape ECs. On the other hand, that the COS is lower in EC vapers compared with TC smokers warrants additional investigation to determine if switching to ECs may form part of a harm-reduction strategy. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT03823885.

Tobacco cigarette (TC) smoking has never been lower in the United States, but electronic cigarette (EC) vaping has reached epidemic proportions among our youth. Endothelial dysfunction, as measured by flow-mediated vasodilation (FMD) is a predictor of future atherosclerosis and adverse cardiovascular events and is impaired in young TC smokers, but whether FMD is also reduced in young EC vapers is uncertain. The aim of this study in otherwise healthy young people was to compare the effects of acute and chronic tobacco cigarette (TC) smoking and electronic cigarette (EC) vaping on FMD. FMD was compared in 47 nonsmokers (NS), 49 chronic EC vapers, and 40 chronic TC smokers at baseline and then after EC vapers (n = 31) and nonsmokers (n = 47) acutely used an EC with nicotine (ECN), EC without nicotine (EC0), and nicotine inhaler (NI) at ~4-wk intervals and after TC smokers (n = 33) acutely smoked a TC, compared with sham control. Mean age (NS, 26.3 ± 5.2 vs. EC, 27.4 ± 5.45 vs. TC, 27.1 ± 5.51 yr, P = 0.53) was similar among the groups, but there were more female nonsmokers. Baseline FMD was not different among the groups (NS, 7.7 ± 4.5 vs. EC:6.6 ± 3.6 vs. TC, 7.9 ± 3.7%∆, P = 0.35), even when compared by group and sex. Acute TC smoking versus control impaired FMD (FMD pre-/postsmoking, -2.52 ± 0.92 vs. 0.65 ± 0.93%∆, P = 0.02). Although the increase in plasma nicotine was similar after EC vapers used the ECN versus TC smokers smoked the TC (5.75 ± 0.74 vs. 5.88 ± 0.69 ng/mL, P = 0.47), acute EC vaping did not impair FMD. In otherwise healthy young people who regularly smoke TCs or ECs, impaired FMD compared with that in nonsmokers was not present at baseline. However, FMD was significantly impaired after smoking one TC, but not after vaping an equivalent "dose" (estimated by change in plasma nicotine) of an EC, consistent with the notion that non-nicotine constituents in TC smoke mediate the impairment. Although it is reassuring that acute EC vaping did not acutely impair FMD, it would be dangerous and premature to conclude that ECs do not lead to atherosclerosis.NEW & NOTEWORTHY In our study of otherwise healthy young people, baseline flow-mediated dilation (FMD), a predictor of atherosclerosis and increased cardiovascular risk, was not different among tobacco cigarette (TC) smokers or electronic cigarette (EC) vapers who had refrained from smoking, compared with nonsmokers. However, acutely smoking one TC impaired FMD in smokers, whereas vaping a similar EC "dose" (as estimated by change in plasma nicotine levels) did not. Finally, although it is reassuring that acute EC vaping did not acutely impair FMD, it would be premature and dangerous to conclude that ECs do not lead to atherosclerosis or increase cardiovascular risk.

Electronic cigarettes (ECs) and tobacco cigarettes (TCs) both release nicotine, a sympathomimetic drug. We hypothesized that baseline heart rate variability (HRV) and hemodynamics would be similar in chronic EC and TC smokers and that after acute EC use, changes in HRV and hemodynamics would be attributable to nicotine, not non-nicotine, constituents in EC aerosol. In 100 smokers, including 58 chronic EC users and 42 TC smokers, baseline HRV and hemodynamics [blood pressure (BP) and heart rate (HR)] were compared. To isolate the acute effects of nicotine vs. non-nicotine constituents in EC aerosol, we compared changes in HRV, BP, and HR in EC users after using an EC with nicotine (ECN), EC without nicotine (EC0), nicotine inhaler (NI), or sham vaping (control). Outcomes were also compared with TC smokers after smoking one TC. Baseline HRV and hemodynamics were not different in chronic EC users and TC smokers. In EC users, BP and HR, but not HRV outcomes, increased only after using the ECN, consistent with a nicotine effect on BP and HR. Similarly, in TC smokers, BP and HR but not HRV outcomes increased after smoking one TC. Despite a similar increase in nicotine, the hemodynamic increases were significantly greater after TC smokers smoked one TC compared with the increases after EC users used the ECN. In conclusion, chronic EC and TC smokers exhibit a similar pattern of baseline HRV. Acute increases in BP and HR in EC users are attributable to nicotine, not non-nicotine, constituents in EC aerosol. The greater acute pressor effects after TC compared with ECN may be attributable to non-nicotine, combusted constituents in TC smoke.NEW & NOTEWORTHY Chronic electronic cigarette (EC) users and tobacco cigarette (TC) smokers exhibit a similar level of sympathetic nerve activity as estimated by heart rate variability. Acute increases in blood pressure (BP) and heart rate in EC users are attribute to nicotine, not non-nicotine, constituents in EC aerosol. Acute TC smoking increased BP significantly more than acute EC use, despite similar increases in plasma nicotine, suggestive of additional adverse vascular effects attributable to combusted, non-nicotine constituents in TC smoke.

Tobacco cigarette smoking is associated with increased sudden death risk, perhaps through adverse effects on ventricular repolarization. The effect of electronic (e-)cigarettes on ventricular repolarization is unknown. The objective of the study was to test the hypothesis that tobacco cigarettes and e-cigarettes have similar adverse effects on electrocardiogram (ECG) indexes of ventricular repolarization and these effects are attributable to nicotine. ECG recordings were obtained in 37 chronic tobacco cigarette smokers, 43 chronic e-cigarette users, and 65 nonusers. Primary outcomes, Tpeak to Tend (Tp-e), Tp-e/QT ratio, and Tp-e/QTc ratio, were measured in tobacco cigarette smokers pre-/post-straw control and smoking one tobacco cigarette and in e-cigarette users and nonusers pre-/post-straw control and using an e-cigarette with and without nicotine (different days). Mean values of the primary outcomes were not different among the three groups at baseline. In chronic tobacco cigarette smokers, all primary outcomes, including the Tp-e (12.9 ± 5.0% vs. 1.5 ± 5%, P = 0.017), Tp-e/QT (14.9 ± 5.0% vs. 0.7 ± 5.1%, P = 0.004), and Tp-e/QTc (11.9 ± 5.0% vs. 2.1 ± 5.1%, P = 0.036), were significantly increased pre-/post-smoking one tobacco cigarette compared with pre-/post-straw control. In chronic e-cigarette users, the Tp-e/QT (6.3 ± 1.9%, P = 0.046) was increased only pre/post using an e-cigarette with nicotine but not pre/post the other exposures. The changes relative to the changes after straw control were greater after smoking the tobacco cigarette compared with using the e-cigarette with nicotine for Tp-e (11.4 ± 4.4% vs. 1.1 ± 2.5%, P < 0.05) and Tp-e/QTc (9.8 ± 4.4% vs. -1.6 ± 2.6%, P = 0.05) but not Tp-e/QT(14.2 ± 4.5% vs. 4.2 ± 2.6%, P = 0.061) . Heart rate increased similarly after the tobacco cigarette and e-cigarette with nicotine. Baseline ECG indexes of ventricular repolarization were not different among chronic tobacco cigarette smokers, electronic cigarette users and nonusers. An adverse effect of acute tobacco cigarette smoking on ECG indexes of ventricular repolarization was confirmed. In chronic e-cigarette users, an adverse effect of using an e-cigarette with nicotine, but not without nicotine, on ECG indexes of ventricular repolarization was also observed.NEW & NOTEWORTHY Abnormal ventricular repolarization, as indicated by prolonged Tpeak-end (Tp-e), is associated with increased sudden death risk. Baseline ECG indexes of repolarization, Tp-e, Tp-e/QT, and Tp-e/QTc, were not different among tobacco cigarette (TC) smokers, electronic cigarette (EC) users, and nonsmokers at baseline, but when TC smokers smoked one TC, all parameters were prolonged. Using an electronic cigarette with nicotine, but not without nicotine, increased the Tp-e/QT. Smoking induces changes in ECG indexes of ventricular repolarization associated with increased sudden death risk.

RNA biology is revolutionized by recent developments of diverse high-throughput technologies for transcriptome-wide profiling of molecular RNA structures. RNA structurome profiling data can be used to identify differentially structured regions between groups of samples. Existing methods are limited in scope to specific technologies and/or do not account for biological variation. Here, we present dStruct which is the first broadly applicable method for differential analysis accounting for biological variation in structurome profiling data. dStruct is compatible with diverse profiling technologies, is validated with experimental data and simulations, and outperforms existing methods.

RNA helicases are a class of enzymes that unwind RNA duplexes in vitro but whose cellular functions are largely enigmatic. Here, we provide evidence that the DEAD-box protein Dbp2 remodels RNA-protein complex (RNP) structure to facilitate efficient termination of transcription in Saccharomyces cerevisiae via the Nrd1-Nab3-Sen1 (NNS) complex. First, we find that loss of DBP2 results in RNA polymerase II accumulation at the 3' ends of small nucleolar RNAs and a subset of mRNAs. In addition, Dbp2 associates with RNA sequence motifs and regions bound by Nrd1 and can promote its recruitment to NNS-targeted regions. Using Structure-seq, we find altered RNA/RNP structures in dbp2∆ cells that correlate with inefficient termination. We also show a positive correlation between the stability of structures in the 3' ends and a requirement for Dbp2 in termination. Taken together, these studies provide a role for RNA remodeling by Dbp2 and further suggests a mechanism whereby RNA structure is exploited for gene regulation.

To study geographic patterns in ophthalmologist supply and patient demand for services in the United States. Google Trends data for the keywords "ophthalmology" and "ophthalmologist" between 2004 and 2019 were queried and normalized to determine relative search volumes (RSV) for each United States state. Ophthalmologist density was calculated by dividing the number of practicing ophthalmologists by the State Census Bureau population estimates. RSV values were divided by ophthalmologist density and normalized to calculate the relative demand index (RDI) for each state. The number of accredited ophthalmology programs per state was acquired through the Accreditation Council for Graduate Medical Education. Ophthalmologist concentration was highly heterogeneous across the country. The states with the highest concentration of ophthalmologist per 10,000 people were Washington, DC (1.42), Maryland (0.94), Massachusetts (0.87), and New York (0.86), while the lowest were Wyoming (0.19), Idaho (0.36), New Mexico (0.38), and Nevada (0.39). RSVs ranged from 36 (Alaska and North Dakota) to 100 (Michigan). The highest RDI was found in South Dakota (100), Delaware (84), Michigan (66), and Arizona (56). The lowest RDI was in Washington, DC (0), Hawaii (7), Oregon (8), and Montana (14). The highest number of ophthalmology residency programs were in New York (18), Texas (9), and California (9), whereas 12 states lacked residency programs altogether. In this study, we found a wide range in the geographic distribution of ophthalmologists and residency programs in the United States. States with the highest relative demand index may represent areas most at risk of unmet medical needs. [ABSTRACT FROM AUTHOR]