Postoperative Cognitive Dysfunction in the Elderly: A Role for Modafinil.

Introductionː Postoperative cognitive dysfunction has long-term consequences of increased mortality, loss of autonomy, and prolonged hospitalization. We sought to determine whether exposing patients to modafinil may attenuate or prevent this devastating syndrome from affecting the elderly postoperatively. Methodsː Adults aged 65 and older and American Society of Anesthesiologists (ASA) I-III physical status scheduled for elective noncardiac/non-neurosurgical surgery were included. Subjects were tested with the Trail Making Test (TMT) and Rey Auditory Visual Learning Test (RAVLT) preoperatively as well as in the immediate postoperative period, at 1 week, and at 3 months. After baseline testing, patients were randomized into three groups: 0) placebo pre and post-procedure; 1) modafinil only pre-procedure and placebo post-procedure; and 2) modafinil pre and post-procedure. A nonsurgical control group was also utilized. Resultsː Seventy-six subjects completed the trial 3 months post-surgery. The baseline RAVLT obtained was analyzed with 2-way ANOVA with repeated measures and showed improvement in learning in all groups (p = 0.03). At 1-week post-surgery, Group 0 subjects demonstrated no learning improvement in the RAVLT. However, there was a significant improvement in learning in both groups that received modafinil (p<0.01), and in the nonsurgical controls (p<0.01). This learning benefit normalized at 3 months. Conclusionː In this prospective, double-blind, placebo-controlled trial, we found that patients who received modafinil showed improvement in the RAVLT at 1 week. However, this learning benefit normalized at 3 months. Further study should examine dose effect, timing, and route of administration to determine if the effect can be enhanced and if in fact, wakefulness is improved post-surgically.

Complications of Transesophageal Echocardiography: A Review of Injuries, Risk Factors, and Management.

Transesophageal echocardiography (TEE) use has become widespread in cardiac surgical operating rooms over the last 2 decades. Surgical and medical decision-making often are guided by the findings of the TEE examination, rendering TEE an invaluable tool both inside and outside the operating room. TEE has become ubiquitous in some parts because it is considered safe and relatively noninvasive. However, it is imperative for clinicians to understand that TEE can cause severe and possibly life-threatening complications, and the risks of TEE must be balanced against its benefits as a diagnostic tool. Upper gastrointestinal (UGI) injuries are the most commonly described complications of TEE; however, the relative infrequency of injuries and lack of uniform reporting make it difficult to definitively identify potential risk factors. Some large retrospective trials suggested that patient factors (age, body mass index, anatomic abnormalities), comorbid conditions (previous stroke), and procedural variables (procedure time, cardiopulmonary bypass time, etc.) are associated with TEE-related injuries. In this narrative review of complications from TEE, the authors focus on the incidence of UGI injuries, the spectrum of injuries associated with TEE, risk factors that may contribute to UGI injuries, as well as diagnosis and management options. Lastly, the discussion focuses on the prevention of injuries as TEE use continues to become more prevalent.

Blunt Trauma to the Heart: A Review of Pathophysiology and Current Management.

Blunt cardiac injury (BCI), defined as an injury to the heart from blunt force trauma, ranges from minor to life-threatening. The majority of BCIs are due to motor vehicle accidents; however, injuries caused by falls, blasts, and sports-related injuries also can be sources of BCI. A significant proportion of patients with BCI do not survive long enough to receive medical care, succumbing to their injuries at the scene of the accident. Additionally, patients with blunt trauma often have coexisting injuries (brain, spine, orthopedic) that can obscure the clinical picture; therefore, a high degree of suspicion often is required to diagnose BCI. Traditionally, hemodynamically stable injuries suspicious for BCI have been evaluated with electrocardiograms and chest radiographs, whereas hemodynamically unstable BCIs have received operative intervention. More recently, computed tomography and echocardiography increasingly have been utilized to identify injuries more rapidly in hemodynamically unstable patients. Transesophageal echocardiography can play an important role in the diagnosis and management of several BCIs that require operative repair. Close communication with the surgical team and access to blood products for potentially massive transfusion also play key roles in maintaining hemodynamic stability. With proper surgical and anesthetic care, survival in cases involving urgent cardiac repair can reach 66%-to-75%. This narrative review focuses on the types of cardiac injuries that are caused by blunt chest trauma, the modalities and techniques currently used to diagnose BCI, and the perioperative management of injuries that require surgical correction.

Assessment of in vitro kinetics and biological impact of nebulized trehalose on human bronchial epithelium.

Trehalose is added in drug formulations to act as fillers or improve aerosolization performance. Its characteristics as a carrier molecule have been explored; however, the fate of trehalose in human airway tissues has not been thoroughly investigated. Here, we investigated the fate of nebulized trehalose using in vitro human air-liquid bronchial epithelial cultures. First, a tracing experiment was conducted using 13C12-trehalose; we measured trehalose distribution in different culture compartments (apical surface liquid, epithelial culture, and basal side medium) at various time points following acute exposure to 13C12-labeled trehalose. We found that 13C12-trehalose was metabolized into 13C6-glucose. The data was then used to model the kinetics of trehalose disappearance from the apical surface of bronchial cultures. Secondly, we evaluated the potential adverse effects of nebulized trehalose on the bronchial cultures after they were acutely exposed to nebulized trehalose up to a level just below its solubility limit (50 g/100 g water). We assessed the ciliary beating frequency and histological characteristics. We found that nebulized trehalose did not lead to marked alteration in ciliary beating frequency and morphology of the epithelial cultures. The in vitro testing approach used here may enable the early selection of excipients for future development of inhalation products.

Discriminating Spontaneous From Cigarette Smoke and THS 2.2 Aerosol Exposure-Related Proliferative Lung Lesions in A/J Mice by Using Gene Expression and Mutation Spectrum Data.

Mice, especially A/J mice, have been widely employed to elucidate the underlying mechanisms of lung tumor formation and progression and to derive human-relevant modes of action. Cigarette smoke (CS) exposure induces tumors in the lungs; but, non-exposed A/J mice will also develop lung tumors spontaneously with age, which raises the question of discriminating CS-related lung tumors from spontaneous ones. However, the challenge is that spontaneous tumors are histologically indistinguishable from the tumors occurring in CS-exposed mice. We conducted an 18-month inhalation study in A/J mice to assess the impact of lifetime exposure to Tobacco Heating System (THS) 2.2 aerosol relative to exposure to 3R4F cigarette smoke (CS) on toxicity and carcinogenicity endpoints. To tackle the above challenge, a 13-gene gene signature was developed based on an independent A/J mouse CS exposure study, following by a one-class classifier development based on the current study. Identifying gene signature in one data set and building classifier in another data set addresses the feature/gene selection bias which is a well-known problem in literature. Applied to data from this study, this gene signature classifier distinguished tumors in CS-exposed animals from spontaneous tumors. Lung tumors from THS 2.2 aerosol-exposed mice were significantly different from those of CS-exposed mice but not from spontaneous tumors. The signature was also applied to human lung adenocarcinoma gene expression data (from The Cancer Genome Atlas) and discriminated cancers in never-smokers from those in ever-smokers, suggesting translatability of our signature genes from mice to humans. A possible application of this gene signature is to discriminate lung cancer patients who may benefit from specific treatments (i.e., EGFR tyrosine kinase inhibitors). Mutational spectra from a subset of samples were also utilized for tumor classification, yielding similar results. "Landscaping" the molecular features of A/J mouse lung tumors highlighted, for the first time, a number of events that are also known to play a role in human lung tumorigenesis, such as Lrp1b mutation and Ros1 overexpression. This study shows that omics and computational tools provide useful means of tumor classification where histopathological evaluation alone may be unsatisfactory to distinguish between age- and exposure-related lung tumors.

Comparison of the biological impact of aerosol of e-vapor device with MESH® technology and cigarette smoke on human bronchial and alveolar cultures.

Exposure to aerosol from electronic vapor (e-vapor) products has been suggested to result in less risk of harm to smokers than cigarette smoke (CS) exposure. Although many studies on e-vapor products have tested the effects of liquid formulations on cell cultures, few have evaluated the effects of aerosolized formulations. We examined the effects of acute exposure to the aerosol of an e-vapor device that uses the MESH® technology (IQOS® MESH, Philip Morris International) and to CS from the 3R4F reference cigarette on human organotypic bronchial epithelial culture and alveolar triculture models. In contrast to 3R4F CS exposure, exposure to the IQOS MESH aerosol (Classic Tobacco flavor) did not cause cytotoxicity in bronchial epithelial cultures or alveolar tricultures despite its greater concentrations of deposited nicotine (3- and 4-fold, respectively). CS exposure caused a marked decrease in the frequency and active area of ciliary beating in bronchial cultures, whereas IQOS MESH aerosol exposure did not. Global mRNA expression and secreted protein profiles revealed a significantly lower impact of IQOS MESH aerosol exposure than 3R4F CS exposure. Overall, our whole aerosol exposure study shows a clearly reduced impact of IQOS MESH aerosol relative to CS in bronchial and alveolar cultures, even at greater nicotine doses.

Respiratory Effects of Exposure to Aerosol From the Candidate Modified-Risk Tobacco Product THS 2.2 in an 18-Month Systems Toxicology Study With A/J Mice.

Smoking cessation is the most effective measure for reducing the risk of smoking-related diseases. However, switching to less harmful products (modified-risk tobacco products [MRTP]) can be an alternative to help reduce the risk for adult smokers who would otherwise continue to smoke. In an 18-month chronic carcinogenicity/toxicity study in A/J mice (OECD Test Guideline 453), we assessed the aerosol of Tobacco Heating System 2.2 (THS 2.2), a candidate MRTP based on the heat-not-burn principle, compared with 3R4F cigarette smoke (CS). To capture toxicity- and disease-relevant mechanisms, we complemented standard toxicology endpoints with in-depth systems toxicology analyses. In this part of our publication series, we report on integrative assessment of the apical and molecular exposure effects on the respiratory tract (nose, larynx, and lungs). Across the respiratory tract, we found changes in inflammatory response following 3R4F CS exposure (eg, antimicrobial peptide response in the nose), with both shared and distinct oxidative and xenobiotic responses. Compared with 3R4F CS, THS 2.2 aerosol exerted far fewer effects on respiratory tract histology, including adaptive tissue changes in nasal and laryngeal epithelium and inflammation and emphysematous changes in the lungs. Integrative analysis of molecular changes confirmed the substantially lower impact of THS 2.2 aerosol than 3R4F CS on toxicologically and disease-relevant molecular processes such as inflammation, oxidative stress responses, and xenobiotic metabolism. In summary, this work exemplifies how apical and molecular endpoints can be combined effectively for toxicology assessment and further supports findings on the reduced respiratory health risks of THS 2.2 aerosol.

Liposomal Bupivacaine Versus Bupivacaine for Intercostal Nerve Blocks in Thoracic Surgery: A Retrospective Analysis.

BACKGROUND: Liposomal bupivacaine (LipoB), delivered via intercostal nerve blocks (ICNBs), is increasingly being used for postoperative pain control in thoracic surgery patients, but there is limited data on its effectiveness when compared to standard bupivacaine. OBJECTIVE: We sought to compare postoperative opioid use, pain control, and length of stay (LOS) in patients undergoing thoracic surgery with LipoB ICNBs vs patients undergoing thoracic surgery with ICNBs using standard bupivacaine. STUDY DESIGN: A retrospective analysis. SETTING: Research took place in a tertiary academic medical center. METHODS: A transition in the standard of care from standard bupivacaine to LipoB for ICNBs in March of 2014 allowed us to compare 2 cohorts: patients who received bupivacaine ICNBs from January 2013 through February of 2014 and patients who received LipoB ICNBs from March 2015 through November 2017. We included patients who underwent thoracic surgery for lung cancer using robotic-assisted thoracic surgery (RATS), video-assisted thoracic surgery (VATS), or traditional open thoracotomy, and documentation of ICNB in the operative note. We collected data on pain scores (Visual Analog Scale [VAS]) and opioid consumption (converted to oral morphine equivalents [OMEs]) intraoperatively, on postoperative day (POD) 0, POD 1, POD 2, and POD 3. We also analyzed data on length of stay [LOS]. A primary analysis was performed on the effects of LipoB vs bupivacaine across all surgery types on opioid consumption, pain scores, and LOS with a secondary analysis on the same endpoints per individual surgery type. RESULTS: A total of 129 patients were included from the predefined study periods (n = 62 LipoB and n = 67 standard bupivacaine). Across all surgery types, LipoB decreased opioid utilization vs standard bupivacaine (P < .01). Post-hoc testing revealed that this difference existed intraoperatively (55 ± 5 vs 69 ± 4 mg OME, P = .03) and on POD 0 (44 ± 6 vs 68 ± 6 mg OME, P < .01). Surgical subtype analysis revealed that this difference was mostly driven by lower opioid consumption in patients undergoing RATS. When compared across all surgery types, LipoB vs bupivacaine did not affect postoperative pain scores. However, subgroup analysis showed that pain scores were lower in the LipoB vs standard bupivacaine group undergoing VATS on POD 0, 1, and 2. The LOS across all thoracic surgery types was lower in the LipoB group when compared to the standard bupivacaine group (median, 4 days [IQR 2.0-6.0] vs median, 5 days [IQR 3.0-8.0], P < .01). Subgroup analysis showed that the LOS in patients undergoing VATS with LipoB ICNBs was shorter compared to patients receiving bupivacaine ICNBs. LIMITATIONS: The retrospective nature of this study makes it prone to several types of bias. CONCLUSION: ICNBs with LipoB for thoracic surgery leads to lower opioid consumption and shorter LOS when compared to ICNBs with standard bupivacaine. The benefit of LipoB over standard bupivacaine for ICNBs appears especially relevant in VATS or RATS procedures. KEY WORDS: Intercostal nerve block, liposomal bupivacaine, RATS, regional anesthesia, robotic-assisted thoracoscopic surgery, thoracotomy, VATS, video-assisted thoracoscopic surgery.

Contemporary Challenges for Fellowship Training in Adult Cardiothoracic Anesthesiology: Perspectives From Program Directors Around the United States.

The fellowship in adult cardiothoracic anesthesiology has matured as an accredited program. This special article addresses current challenges in this educational milieu. The first challenge relates to serving as a program director in the contemporary era. The second challenge deals with the accreditation process, including the site visit. The third challenge discusses the integration of structural heart disease and interventional echocardiography into daily practice. The fourth challenge deals with the issues that face fellowship education in the near future. Taken together, these perspectives provide a review of the contemporary challenges facing fellowship education in adult cardiothoracic anesthesiology.

Comparison of the basic morphology and function of 3D lung epithelial cultures derived from several donors.

In vitro models of the human lung play an essential role in evaluating the toxicity of inhaled compounds and understanding the development of respiratory diseases. Three-dimensional (3D) organotypic models derived from lung basal epithelial cells and grown at the air-liquid interface resemble human airway epithelium in multiple aspects, including morphology, cell composition, transcriptional profile, and xenobiotic metabolism. Whether the different characteristics of basal cell donors have an impact on model characteristics and responses remains unknown. In addition, studies are often conducted with 3D cultures from one donor, assuming a representative response on the population level. Whether this assumption is correct requires further investigation. In this study, we compared the morphology and functionality of 3D organotypic bronchial and small airway cultures from different donors at different weeks after air-lift to assess the interdonor variability in these parameters. The thickness, cell type composition, and transepithelial electrical resistance varied among the donors and over time after air-lift. Cilia beating frequency increased in response to isoproterenol treatment in both culture types, independent of the donor. The cultures presented low basal cytochrome P450 (CYP) 1A1/1B1 activity, but 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment induced CYP1A1/1B1 activity regardless of the donor. In conclusion, lung epithelial cultures prepared from different donors present diverse morphology but similar functionality and metabolic activity, with certain variability in their response to stimulation.

Structural, functional, and molecular impact on the cardiovascular system in ApoE-/- mice exposed to aerosol from candidate modified risk tobacco products, Carbon Heated Tobacco Product 1.2 and Tobacco Heating System 2.2, compared with cigarette smoke.

AIM: To investigate the molecular, structural, and functional impact of aerosols from candidate modified risk tobacco products (cMRTP), the Carbon Heated Tobacco Product (CHTP) 1.2 and Tobacco Heating System (THS) 2.2, compared with that of mainstream cigarette smoke (CS) on the cardiovascular system of ApoE-/- mice. METHODS: Female ApoE-/- mice were exposed to aerosols from THS 2.2 and CHTP 1.2 or to CS from the 3R4F reference cigarette for up to 6 months at matching nicotine concentrations. A Cessation and a Switching group (3 months exposure to 3R4F CS followed by filtered air or CHTP 1.2 for 3 months) were included. Cardiovascular effects were investigated by echocardiographic, histopathological, immunohistochemical, and transcriptomics analyses. RESULTS: Continuous exposure to cMRTP aerosols did not affect atherosclerosis progression, heart function, left ventricular (LV) structure, or the cardiovascular transcriptome. Exposure to 3R4F CS triggered atherosclerosis progression, reduced systolic ejection fraction and fractional shortening, caused heart LV hypertrophy, and initiated significant dysregulation in the transcriptomes of the heart ventricle and thoracic aorta. Importantly, the structural, functional, and molecular changes caused by 3R4F CS were improved in the smoking cessation and switching groups. CONCLUSION: Exposure to cMRTP aerosols lacked most of the CS exposure-related functional, structural, and molecular effects. Smoking cessation or switching to CHTP 1.2 aerosol caused similar recovery from the 3R4F CS effects in the ApoE-/- model, with no further acceleration of plaque progression beyond the aging-related rate.

Application of a multi-layer systems toxicology framework for in vitro assessment of the biological effects of Classic Tobacco e-liquid and its corresponding aerosol using an e-cigarette device with MESH™ technology.

We previously proposed a systems toxicology framework for in vitro assessment of e-liquids. The framework starts with the first layer aimed at screening the potential toxicity of e-liquids, followed by the second layer aimed at investigating the toxicity-related mechanism of e-liquids, and finally, the third layer aimed at evaluating the toxicity-related mechanism of the corresponding aerosols. In this work, we applied this framework to assess the impact of the e-liquid MESH Classic Tobacco and its aerosol compared with that of cigarette smoke (CS) from the 3R4F reference cigarette. In the first layer, we evaluated the cytotoxicity profile of the MESH Classic Tobacco e-liquid (containing humectants, nicotine, and flavors) and its Base e-liquid (containing humectant and nicotine only) in comparison with total particulate matter (TPM) of 3R4F CS using primary bronchial epithelial cell cultures. In the second layer, the same culture model was used to explore changes in specific markers using high-content screening assays to identify potential toxicity-related mechanisms induced by the MESH Classic Tobacco and Base e-liquids beyond cell viability in comparison with the 3R4F CS TPM-induced effects. Finally, in the third layer, we compared the impact of exposure to the MESH Classic Tobacco or Base aerosols with 3R4F CS using human organotypic air-liquid interface buccal and small airway epithelial cultures. The results showed that the cytotoxicity of the MESH Classic Tobacco liquid was similar to the Base liquid but lower than 3R4F CS TPM at comparable nicotine concentrations. Relative to 3R4F CS exposure, MESH Classic Tobacco aerosol exposure did not cause tissue damage and elicited lower changes in the mRNA, microRNA, and protein markers. In the context of tobacco harm reduction strategy, the framework is suitable to assess the potential-reduced impact of electronic cigarette aerosol relative to CS.

A lower impact of an acute exposure to electronic cigarette aerosols than to cigarette smoke in human organotypic buccal and small airway cultures was demonstrated using systems toxicology assessment.

In the context of tobacco harm-reduction strategy, the potential reduced impact of electronic cigarette (EC) exposure should be evaluated relative to the impact of cigarette smoke exposure. We conducted a series of in vitro studies to compare the biological impact of an acute exposure to aerosols of "test mix" (flavors, nicotine, and humectants), "base" (nicotine and humectants), and "carrier" (humectants) formulations using MarkTen® EC devices with the impact of exposure to smoke of 3R4F reference cigarettes, at a matching puff number, using human organotypic air-liquid interface buccal and small airway cultures. We measured the concentrations of nicotine and carbonyls deposited in the exposure chamber after each exposure experiment. The deposited carbonyl concentrations were used as representative measures to assess the reduced exposure to potentially toxic volatile substances. We followed a systems toxicology approach whereby functional biological endpoints, such as histopathology and ciliary beating frequency, were complemented by multiplex and omics assays to measure secreted inflammatory proteins and whole-genome transcriptomes, respectively. Among the endpoints analyzed, the only parameters that showed a significant response to EC exposure were secretion of proteins and whole-genome transcriptomes. Based on the multiplex and omics analyzes, the cellular responses to EC aerosol exposure were tissue type-specific; however, those alterations were much smaller than those following cigarette smoke exposure, even when the EC aerosol exposure under the testing conditions resulted in a deposited nicotine concentration approximately 200 times that in saliva of EC users.

Assessment of a 72-hour repeated exposure to Swedish snus extract and total particulate matter from 3R4F cigarette smoke on gingival organotypic cultures.

Swedish snus is a smokeless tobacco product that contains reduced levels of harmful compounds compared with cigarette smoke. In Sweden, where snus use exceeds smoking among men, relatively low rates of major smoking-related diseases have been recorded. To better understand how snus use could align with current tobacco harm reduction strategies, its potential mechanisms of toxicity must be investigated. This study aimed to determine, via a systems toxicology approach, the biological impact of repeated 72-hour exposure of human gingival epithelial organotypic cultures to extracts from both a commercial and a reference snus and the total particulate matter (TPM) from cigarette smoke. At concentrations relevant for human use, cultures treated with snus extracts induced mild, generally reversible biological changes, while TPM treatment induced substantial morphological and inflammatory alterations. Network enrichment analysis and integrative analysis of the global mRNA and miRNA expression profiles indicated a limited and mostly transient impact of the snus extracts, in particular on xenobiotic metabolism, while the effects of TPM were marked and sustained over time. High-confidence miRNAs that might be related to pathological conditions in vivo were identified. This study highlights the limited biological impact of Swedish snus extract on human organotypic gingival cultures.

A lung/liver-on-a-chip platform for acute and chronic toxicity studies.

The merging of three-dimensional in vitro models with multi-organ-on-a-chip (MOC) technology has taken in vitro assessment of chemicals to an unprecedented level. By connecting multiple organotypic models, MOC allows for the crosstalk between different organs to be studied to evaluate a compound's safety and efficacy better than with single cultures. The technology could also improve the toxicological assessment of aerosols that have been implicated in the development of chronic obstructive pulmonary disease, asthma, or lung cancer. Here we report the development of a lung/liver-on-a-chip, connecting in a single circuit, normal human bronchial epithelial (NHBE) cells cultured at the air-liquid interface (ALI), and HepaRG™ liver spheroids. Maintenance of the individual tissues in the chip increased NHBE ALI tissue transepithelial electrical resistance and decreased HepaRG™ spheroid adenosine triphosphate content as well as cytochrome P450 (CYP) 1A1/1B1 inducibility. CYP inducibility was partly restored when HepaRG™ spheroids were cocultured with NHBE ALI tissues. Both tissues remained viable and functional for 28 days when cocultured in the chip. The capacity of the HepaRG™ spheroids to metabolize compounds present in the medium and to modulate their toxicity was proven using aflatoxin B1 (AFB1). AFB1 toxicity in NHBE ALI tissues decreased when HepaRG™ spheroids were present in the same chip circuit, proving that the HepaRG™-mediated detoxification is protecting/decreasing from AFB1-mediated cytotoxicity. The lung/liver-on-a-chip platform presented here offers new opportunities to study the toxicity of inhaled aerosols or to demonstrate the safety and efficacy of new drug candidates targeting the human lung.

Assessment of the impact of aerosol from a potential modified risk tobacco product compared with cigarette smoke on human organotypic oral epithelial cultures under different exposure regimens.

Cigarette smoke (CS) is affecting considerably the oral mucosa. Heating, instead of burning, tobacco reduces consistently the amount of toxic compounds and may exert a lower impact on oral health than combusted cigarettes. The carbon-heated tobacco product 1.2 (CHTP1.2) is a potential modified risk tobacco product (MRTP) based on heat-not-burn technology. Using a systems toxicology assessment framework, we compared the effects of exposure to CHTP1.2 aerosol with those of CS from a reference cigarette (3R4F). Human organotypic cultures derived from buccal and gingival epithelia were exposed acutely (28-min) or repeatedly (28 min/day for 3 days), respectively, to two matching concentrations of CHTP1.2 aerosol or 3R4F CS, and a non-diluted (100%) CHTP1.2 aerosol. The results showed an absence of cytotoxicity, reduction in pathophysiological alterations, toxicological marker proteins, and inflammatory mediators following exposure to CHTP1.2 aerosol compared with 3R4F CS. Changes in mRNA and miRNA expression were linked by an integrative analysis approach, suggesting a regulatory role of miRNAs in several smoke/disease-relevant biological processes induced by 3R4F CS. The identification of mechanisms by which potential MRTPs can reduce the impact of tobacco use on biological systems is of great importance in understanding the molecular basis of the smoking harm reduction paradigm.