Determination of chemical constituent yields in e-cigarette aerosol using partial and whole pod collections, a comparative analysis.

Literature reports the chemical constituent yields of electronic nicotine delivery systems (ENDS) aerosol collected using a range of aerosol collection strategies. The number of puffs to deplete an ENDS product varies widely, but collections often consist of data from the first 50-100 puffs. However, it is not clear whether these discrete puff blocks are representative of constituent yields over the life of a pod. We aimed to assess the effect of differing aerosol collection strategies on reported yields for select chemical constituents in the aerosol of closed pod-based ENDS products. Constituents analyzed were chosen to reflect important classes of compounds from the Final Premarket Tobacco Product Application Guidance. Yields were normalized to total device mass loss (DML). Collection strategies that consisted of partial pod collection were valid for determining yields of constituents whose DML normalized yields were consistent for the duration of pod life. These included primary aerosol constituents, such as propylene glycol, glycerol, and nicotine, and whole pod yields could be determined from initial puff blocks. However, changes were observed in the yields of some metals, some carbonyl compounds, and glycidol over pod life in a chemical constituent and product dependent manner. These results suggest that collection strategies consisting of initial puff block collections require validation per chemical constituent/product and are not appropriate for chemical constituents with variable yields over pod life. Whole pod collection increased sensitivity and accuracy in determining metal, carbonyl, and glycidol yields compared to puff block-based collection methodologies for all products tested.

Microtubule disruption reduces metastasis more effectively than primary tumor growth.

Clinical cancer imaging focuses on tumor growth rather than metastatic phenotypes. The microtubule-depolymerizing drug, Vinorelbine, reduced the metastatic phenotypes of microtentacles, reattachment and tumor cell clustering more than tumor cell viability. Treating mice with Vinorelbine for only 24 h had no significant effect on primary tumor survival, but median metastatic tumor survival was extended from 8 to 30 weeks. Microtentacle inhibition by Vinorelbine was also detectable within 1 h, using tumor cells isolated from blood samples. As few as 11 tumor cells were sufficient to yield 90% power to detect this 1 h Vinorelbine drug response, demonstrating feasibility with the small number of tumor cells available from patient biopsies. This study establishes a proof-of-concept that targeted microtubule disruption can selectively inhibit metastasis and reveals that existing FDA-approved therapies could have anti-metastatic actions that are currently overlooked when focusing exclusively on tumor growth.

Prediction of potential passive exposure from commercial electronic nicotine delivery systems using exhaled breath analysis and computational fluid dynamic techniques.

Use of computational fluid dynamic (CFD) modeling to predict temporal and spatial constituent exposure for non-electronic nicotine delivery systems (ENDS) users (passive exposure) provides a more efficient methodology compared to conducting actual exposure studies. We conducted a clinical study measuring exhaled breath concentrations of glycerin, propylene glycol, nicotine, benzoic acid, formaldehyde, acetaldehyde, acrolein, menthol and carbon monoxide from use of eight different commercial ENDS devices and a non-menthol and menthol cigarette. Because baseline adjusted levels of other constituents were not consistently above the limit of detection, the mean minimum and maximum per puff exhaled breath concentrations (N= 20/product) of glycerin (158.7-260.9µg), propylene glycol (0.941-3.58µg), nicotine (0.10-1.06µg), and menthol (0.432-0.605µg) from use of the ENDS products were used as input parameters to predict temporal and spatial concentrations in an environmental chamber, office, restaurant, and car using different ENDS use scenarios. Among these indoor locations and ENDS use scenarios, the car with closed windows resulted in the greatest concentrations while opening the car windows produced the lowest concentrations. The CFD predicted average maximum glycerin and propylene glycol concentration ranged from 0.25 to 1068µg m-3and 1.5 pg m-3to 13.56µg m-3,respectively. For nicotine and menthol the CFD predicted maximum concentration ranged from 0.16 pg m-3to 4.02µg m-3and 0.068 pg m-3to 2.43µg m-3, respectively. There was better agreement for CFD-predicted nicotine concentrations than glycerin and propylene glycol with published reports highlighting important experimental and computational variables. Maximum measured nicotine levels from environmental tobacco smoke in offices, restaurants, and cars exceeded our maximum average CFD predictions by 7-97 times. For all the measured exhaled breath constituents and CFD predicted constituents, except for propylene glycol and glycerin, concentrations were less from use of ENDS products compared to combustible cigarettes. NCT number: NCT04143256.

Changes in Biomarkers of Cigarette Smoke Exposure After 6 Days of Switching Exclusively or Partially to Use of the JUUL System with Two Nicotine Concentrations: A Randomized Controlled Confinement Study in Adult Smokers.

INTRODUCTION: Evidence suggests that cigarette smokers who switch to electronic nicotine delivery systems (ENDS) reduce their exposure to harmful toxicants and carcinogens. It is unclear if dual-use is associated with decreases in exposure to toxicants. METHODS: This parallel-group confinement study assessed changes in biomarkers of exposure (BOEs) over six days among healthy adult smokers who were randomized into 1 of 11 study groups: eight JUUL-brand System (JUUL) groups (4 JUUL flavors [Virginia Tobacco, Menthol, Mint, Mango] × 2 nicotine concentrations [5.0% or 3.0% by weight]); Dual-Use group used preferred JUUL flavor (5.0% nicotine) and ≤50% usual brand (UB) cigarettes/day; UB Cigarette group and one group abstained from all tobacco/nicotine product use (Abstinence group). Urine and blood analysis assessed changes in primary BOE endpoints (NNAL, 3-HPMA, MHBMA, S-PMA COHb) and secondary BOE endpoints (NNN, HMPMA, CEMA, 1-OHP, O-toluidine, 2-NA, 4-ABP) among 279 adult smokers. RESULTS: In JUUL groups, median percent reductions in primary BOEs (Day 6-Baseline) were 90%-≥100% of Abstinence; there were no significant differences between JUUL groups and Abstinence. All reductions in JUUL groups were substantially and statistically significantly greater than reductions in the UB Cigarette group (ps < 0.025). Median reductions in primary BOEs in the Dual-Use group were 43%-55% of Abstinence. Similar results were observed for secondary BOEs. CONCLUSION: This study suggests that the use of JUUL as a complete or partial substitute (i.e., dual-use with ≥50% reduction in cigarette consumption) for combustible cigarettes can substantially reduce exposure to multiple toxins associated with cigarette smoking. IMPLICATIONS: This study adds to the growing body of evidence supporting the utility of ENDS products as potentially reduced-harm alternatives to cigarettes for adult smokers. Adult smokers who switched completely from cigarette smoking to use of the JUUL System ("JUUL") in two nicotine concentrations (5.0% and 3.0%) and four flavors significantly reduced their exposure to multiple classes of cigarette-related toxicants. Additionally, smokers who used JUUL and continued smoking but reduced their daily cigarette consumption by ≥50% (dual users) also significantly reduced their toxicant exposure compared to cigarette smoking.

Lipid tethering of breast tumor cells reduces cell aggregation during mammosphere formation.

Mammosphere assays are widely used in vitro to identify prospective cancer-initiating stem cells that can propagate clonally to form spheres in free-floating conditions. However, the traditional mammosphere assay inevitably introduces cell aggregation that interferes with the measurement of true mammosphere forming efficiency. We developed a method to reduce tumor cell aggregation and increase the probability that the observed mammospheres formed are clonal in origin. Tethering individual tumor cells to lipid anchors prevents cell drift while maintaining free-floating characteristics. This enables real-time monitoring of single tumor cells as they divide to form mammospheres. Monitoring tethered breast cancer cells provided detailed size information that correlates directly to previously published single cell tracking data. We observed that 71% of the Day 7 spheres in lipid-coated wells were between 50 and 150 µm compared to only 37% in traditional low attachment plates. When an equal mixture of MCF7-GFP and MCF7-mCherry cells were seeded, 65% of the mammospheres in lipid-coated wells demonstrated single color expression whereas only 32% were single-colored in low attachment wells. These results indicate that using lipid tethering for mammosphere growth assays can reduce the confounding factor of cell aggregation and increase the formation of clonal mammospheres.

Mechanoactivation of NOX2-generated ROS elicits persistent TRPM8 Ca2+ signals that are inhibited by oncogenic KRas.

Changes in the mechanical microenvironment and mechanical signals are observed during tumor progression, malignant transformation, and metastasis. In this context, understanding the molecular details of mechanotransduction signaling may provide unique therapeutic targets. Here, we report that normal breast epithelial cells are mechanically sensitive, responding to transient mechanical stimuli through a two-part calcium signaling mechanism. We observed an immediate, robust rise in intracellular calcium (within seconds) followed by a persistent extracellular calcium influx (up to 30 min). This persistent calcium was sustained via microtubule-dependent mechanoactivation of NADPH oxidase 2 (NOX2)-generated reactive oxygen species (ROS), which acted on transient receptor potential cation channel subfamily M member 8 (TRPM8) channels to prolong calcium signaling. In contrast, the introduction of a constitutively active oncogenic KRas mutation inhibited the magnitude of initial calcium signaling and severely blunted persistent calcium influx. The identification that oncogenic KRas suppresses mechanically-induced calcium at the level of ROS provides a mechanism for how KRas could alter cell responses to tumor microenvironment mechanics and may reveal chemotherapeutic targets for cancer. Moreover, we find that expression changes in both NOX2 and TRPM8 mRNA predict poor clinical outcome in estrogen receptor (ER)-negative breast cancer patients, a population with limited available treatment options. The clinical and mechanistic data demonstrating disruption of this mechanically-activated calcium pathway in breast cancer patients and by KRas activation reveal signaling alterations that could influence cancer cell responses to the tumor mechanical microenvironment and impact patient survival.

The American College of Surgeons Response to the COVID-19 Pandemic (Part II) : Advocacy and Public Policy.

From the onset of the COVID-19 global pandemic of 2020, the American College of Surgeons (ACS) has been a leader in disseminating credible information on the clinical and scientific aspects of the disease. As governmental regulations enforced the closure of hospitals and operating rooms to elective surgical cases as part of its "shelter-in-place" public lockdown policies, the ACS brought specialty societies together to create guidelines to protect patients and preserve surgical quality. Federal agencies made available financial aid programs to mitigate the economic impact of the outbreak. The division of advocacy and health policy of the ACS made certain that the interests of surgeons and their patients were served. Steven Wexner, member of the Board of Regents of the ACS interviewed the medical directors of the division, Frank Opelka in quality and health policy, and Patrick Bailey in advocacy, for their stories of how the College responded to the many health and public policy issues that came before Congress and governmental agencies during the pandemic.

Insights on CTC Biology and Clinical Impact Emerging from Advances in Capture Technology.

Circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) have been shown to correlate negatively with patient survival. Actual CTC counts before and after treatment can be used to aid in the prognosis of patient outcomes. The presence of circulating tumor materials (CTMat) can advertise the presence of metastasis before clinical presentation, enabling the early detection of relapse. Importantly, emerging evidence is indicating that cancer treatments can actually increase the incidence of CTCs and metastasis in pre-clinical models. Subsequently, the study of CTCs, their biology and function are of vital importance. Emerging technologies for the capture of CTC/CTMs and CTMat are elucidating vitally important biological and functional information that can lead to important alterations in how therapies are administered. This paves the way for the development of a "liquid biopsy" where treatment decisions can be informed by information gleaned from tumor cells and tumor cell debris in the blood.

Single-Cell Tracking of Breast Cancer Cells Enables Prediction of Sphere Formation from Early Cell Divisions.

The mammosphere assay has become widely employed to quantify stem-like cells in a population. However, the problem is there is no standard protocol employed by the field. Cell seeding densities of 1,000 to 100,000 cells/mL have been reported. These high densities lead to cellular aggregation. To address this, we have individually tracked 1,127 single MCF-7 and 696 single T47D human breast tumor cells by eye over the course of 14 days. This tracking has given us detailed information for the commonly used endpoints of 5, 7, and 14 days that is unclouded by cellular aggregation. This includes mean sphere sizes, sphere-forming efficiencies, and a well-defined minimum size for both lines. Importantly, we have correlated early cell division with eventual sphere formation. At 24 hr post seeding, we can predict the total spheres on day 14 with 98% accuracy in both lines. This approach removes cell aggregation and potentially shortens a 5- to 14-day assay to a 24 hours.

Thiodipeptides targeting the intestinal oligopeptide transporter as a general approach to improving oral drug delivery.

The broad substrate capacity of the intestinal oligopeptide transporter, PepT1, has made it a key target of research into drug delivery. Whilst the substrate capacity of this transporter is broad, studies have largely been limited to small peptides and peptide-like drugs. Here, we demonstrate for the first time that a diverse range of drugs can be targeted towards transport by PepT1 using a hydrolysis resistant carrier. Eleven prodrugs were synthesized by conjugating modified dipeptides containing a thioamide bond to the approved drugs ibuprofen, gabapentin, propofol, aspirin, acyclovir, nabumetone, atenolol, zanamivir, baclofen and mycophenolate. Except for the aspirin and acyclovir prodrugs, which were unstable in the assay conditions and were not further studied, the prodrugs were tested for affinity and transport by PepT1 expressed in Xenopus laevis oocytes: binding affinities ranged from approximately 0.1 to 2 mM. Compounds which showed robust transport in an oocyte trans-stimulation assay were then tested for transcellular transport in Caco-2 cell monolayers: all five tested prodrugs showed significant PepT1-mediated transcellular uptake. Finally, the ibuprofen and propofol prodrugs were tested for absorption in rats: following oral dosing the intact prodrugs and free ibuprofen were measured in the plasma. This provides proof-of-concept for the idea of targeting poorly bioavailable drugs towards PepT1 transport as a general means of improving oral permeability.

Real-time scratch assay reveals mechanisms of early calcium signaling in breast cancer cells in response to wounding.

Aggressive cellular phenotypes such as uncontrolled proliferation and increased migration capacity engender cellular transformation, malignancy and metastasis. While genetic mutations are undisputed drivers of cancer initiation and progression, it is increasingly accepted that external factors are also playing a major role. Two recently studied modulators of breast cancer are changes in the cellular mechanical microenvironment and alterations in calcium homeostasis. While many studies investigate these factors separately in breast cancer cells, very few do so in combination. This current work sets a foundation to explore mechano-calcium relationships driving malignant progression in breast cancer. Utilizing real-time imaging of an in vitro scratch assay, we were able to resolve mechanically-sensitive calcium signaling in human breast cancer cells. We observed rapid initiation of intracellular calcium elevations within seconds in cells at the immediate wound edge, followed by a time-dependent increase in calcium in cells at distances up to 500µm from the scratch wound. Calcium signaling to neighboring cells away from the wound edge returned to baseline within seconds. Calcium elevations at the wound edge however, persisted for up to 50 minutes. Rigorous quantification showed that extracellular calcium was necessary for persistent calcium elevation at the wound edge, but intercellular signal propagation was dependent on internal calcium stores. In addition, intercellular signaling required extracellular ATP and activation of P2Y2 receptors. Through comparison of scratch-induced signaling from multiple cell lines, we report drastic reductions in response from aggressively tumorigenic and metastatic cells. The real-time scratch assay established here provides quantitative data on the molecular mechanisms that support rapid scratch-induced calcium signaling in breast cancer cells. These mechanisms now provide a clear framework for investigating which short-term calcium signals promote long-term changes in cancer cell biology.

Impact of an integrated intervention program on atorvastatin adherence: a randomized controlled trial.

BACKGROUND: This trial evaluated the effectiveness of an integrated intervention program that included a 3-to-5-minute nurse counseling session, copay relief cards, and a monthly newsletter on adherence to atorvastatin treatment. METHODS AND RESULTS: A prospective, integrated (composed of nurse counseling, adherence tip sheet, copay relief card, opportunity to enroll in 12-week cholesterol management program) randomized interventional study was designed involving patients >21 years of age who were prescribed atorvastatin at a large single-specialty cardiovascular physician practice in Illinois from March 2010 to May 2011. Data from the practice's electronic medical record were matched/merged to IMS Health's longitudinal data. A total of 500 patients were enrolled (125 in the control arm; 375 in the intervention arm). After data linkage, 53 control patients and 155 intervention patients were included in the analysis. RESULTS: Mean age was 67.8 years (control) and 69.5 years (intervention); 67.9% and 58.7%, respectively, were male. The mean 6-month adherence rate was 0.82 in both arms. The mean proportion of days covered for both the new-user control and intervention groups was the same, averaging 0.70 day (standard deviation [SD], 0.27 day); for continuing users, the proportion of days covered for the control group was 0.83 (SD, 0.24) and for the intervention group was 0.84 (SD, 0.22). For continuing users, the control group had mean persistent days of 151.6 (SD, 50.2) compared with 150.9 days (SD, 50.9) for the intervention group. New users had fewer persistent days (control 111.4 days, SD, 69.6 days; intervention 112.0 days, SD, 58.8 days) compared with continuing users. The Cox proportional hazards model of the risk of discontinuation with index therapy was not significantly different between the intervention and control groups (hazard ratio 0.83, P = 0.55). CONCLUSION: The integrated intervention program did not significantly improve atorvastatin adherence relative to usual care in the studied patient population.

Bioavailability through PepT1: the role of computer modelling in intelligent drug design.

In addition to being responsible for the majority of absorption of dietary nitrogen, the mammalian proton-coupled di- and tri-peptide transporter PepT1 is also recognised as a major route of drug delivery for several important classes of compound, including beta-lactam antibiotics and angiotensin-converting enzyme inhibitors. Thus there is considerable interest in the PepT1 protein and especially its substrate binding site. In the absence of a crystal structure, computer modelling has been used to try to understand the relationship between PepT1 3D structure and function. Two basic approaches have been taken: modelling the transporter protein, and modelling the substrate. For the former, computer modelling has evolved from early interpretations of the twelve transmembrane domain structure to more recent homology modelling based on recently crystallised bacterial members of the major facilitator superfamily (MFS). Substrate modelling has involved the proposal of a substrate binding template, to which all substrates must conform and from which the affinity of a substrate can be estimated relatively accurately, and identification of points of potential interaction of the substrate with the protein by developing a pharmacophore model of the substrates. Most recently, these two approaches have moved closer together, with the attempted docking of a substrate library onto a homology model of the human PepT1 protein. This article will review these two approaches in which computers have been applied to peptide transport and suggest how such computer modelling could affect drug design and delivery through PepT1.

The in vitro transport of model thiodipeptide prodrugs designed to target the intestinal oligopeptide transporter, PepT1.

A thiodipeptide carrier system is shown to be effective at enabling a range of covalently bound molecules, including benzyl, benzoyl and ibuprofen conjugates, to be transported via the intestinal peptide transporter PepT1, demonstrating its potential as a rational drug delivery target.