Revisiting Exome Data Identified Missed Splice Site Variant of the Asparagine Synthetase ( ASNS ) Gene.

Next-generation sequencing, such as whole-exome sequencing (WES), is increasingly used in the study of Mendelian disorders, yet many are reported as "negative." Inappropriate variant annotation and filtering steps are reasons for missing the molecular diagnosis. Noncoding variants, including splicing mutations, are examples of variants that can be overlooked. Herein, we report a family of four affected newborns, and all presented with severe congenital microcephaly. Initial research WES analysis identified a damaging homozygous variant in NME1 gene as a possible cause of primary microcephaly phenotype in these patients. However, reanalysis of the exome data uncovered a biallelic splice site variant in asparagine synthetase gene which seems to be the possible cause of the phenotype in these patients. This study highlights the importance of revisiting the exome data and the issue of "negative" exome and the afterward approaches to identify and prove new candidate genes.

Elevated oxidative stress biomarkers in adults with segmented sleep patterns.

STUDY OBJECTIVES: To investigate the association between different sleep patterns and inflammatory and oxidative stress biomarkers in adults. METHODS: A total of 321 consented adults who fulfilled the inclusion criteria were recruited in this cross sectional study. The inclusion criteria were mainly based on age of 18-59 years of apparently healthy adults. To identify sleep patterns, participants were requested to wear the actigraphy for one week for 24- hours a day. Fasting blood was collected from each participant at day 8. The blood serum was analyzed for inflammatory and oxidative stress biomarkers. Sleep patterns were defined as monophasic (one episode of night sleep) bi-phasic (two episodes of sleep; night and afternoon siesta) and polyphasic sleep pattern (three or more sleep episodes). RESULTS: There was no correlation between night sleep duration, total sleep in 24-hours and napping with inflammatory and oxidative stress biomarkers; high-sensitivity C-reactive protein (hs-CRP), malondialdehyde (MDA), total glutathione (GSH) and basal oxidisability status (BOS). Actigraphy reports showed three sleep patterns in this cohort, monophasic (24.3%), biphasic-napping (45.2%) and polyphasic (30.5%). Individuals with segmented sleep patterns were significantly associated with oxidative stress biomarkers. Polyphasic sleep pattern was significantly associated with higher BOS (P-value =0.023), while biphasic sleep pattern showed higher MDA (P-value=0.036) as compared to monophasic sleep pattern. Total GSH was significantly higher in monophasic sleepers (P-value =0.046). There was no difference in serum hs-CRP among all sleep patterns. CONCLUSIONS: Segmented sleep in polyphasic and biphasic sleep patterns are associated with higher serum MDA and BOS in particular. Further studies are recommended on the cardiometabolic impact of oxidative stress biomarkers in individual with segmented sleep.

Base of the tongue hyalinizing clear cell carcinoma: Case report and literature review.

Hyalinizing clear cell carcinoma is an uncommon neoplasm arising in minor salivary glands. We present a rare case of hyalinizing clear cell carcinoma in the base of the tongue. We report a case of a 38-year-old female presented with a progressive history of hemoptysis and dysphagia over the course of 4 years. Examination revealed a mass originating from the base of the tongue with a biopsy confirmed as hyalinizing clear cell carcinoma . An Ovid MEDLINE and PubMed literature review was conducted due to the rarity of this type of tumor. The patient underwent surgical excision with immediate reconstruction with radial forearm free flap followed with adjuvant radiotherapy and was disease free at her most recent follow-up (12 months). Our review included a total of 13 new cases, including our case. The majority of the cases presented with dysphagia. Surgical excision is the mainstay of treatment, and overall these patients have a good prognosis. Our case highlights a rare presentation of hyalinizing clear cell carcinoma of the base of the tongue, successfully treated with surgical excision, free tissue reconstruction and adjuvant radiotherapy.

Cytotoxicity, mutagenicity and genotoxicity of electronic cigarettes emission aerosols compared to cigarette smoke: the REPLICA project.

Concerns have recently increased that the integrity of some scientific research is questionable due to the inability to reproduce the claimed results of some experiments and thereby confirm that the original researcher's conclusions were justified. This phenomenon has been described as 'reproducibility crisis' and affects various fields from medicine to basic applied sciences. In this context, the REPLICA project aims to replicate previously conducted in vitro studies on the toxicity of cigarette smoke and e-cigarette aerosol, sometimes adding experiments or conditions where necessary, in order to verify the robustness and replicability of the data. In this work the REPLICA Team replicated biological and toxicological assessment published by Rudd and colleagues in 2020. As in the original paper, we performed Neutral Red Uptake (NRU) assay for the evaluation of cytotoxicity, Ames test for the evaluation of mutagenesis and In Vitro Micronuclei (IVMN) assay for the evaluation of genotoxicity on cells treated with cigarette smoke or e-cigarette aerosol. The results showed high cytotoxicity, mutagenicity and genotoxicity induced by cigarette smoke, but slight or no cytotoxic, mutagenic and genotoxic effects induced by the e-cigarette aerosol. Although the two studies presented some methodological differences, the findings supported those previously presented by Rudd and colleagues.

Reference Ranges of Serum Anti-Müllerian Hormone in Healthy Reproductive-aged Omani Women.

Objectives: Anti-Müllerian hormone (AMH), a glycoprotein that belongs to the transforming growth factor-beta superfamily, is important for women's health. We aimed to determine the age-specific reference range of serum AMH in healthy Omani women from reproductive ages to menopause. Methods: This cross-sectional cohort study was conducted among a group of healthy 20-50 years old Omani women. The participants were required to have body mass index < 32 kg/m2, regular periods, no history of chronic illness, polycystic ovary syndrome, or gynecological operation. They were also required to not be using any hormonal contraceptive. Serum concentrations of AMH, follicle-stimulating hormone, luteinizing hormone, progesterone, and hemoglobin A1c were measured. AMH-age nomogram and AMH levels were compared between the six selected age groups. Results: The subjects were 319 Omani women aged 20-50 years. Serum AMH concentrations were found to decrease progressively with increasing age. An exponential model defined as √AMH = 479.02 × 0.91age was selected to explain the reduction in AMH with age (R2 = 0.298). The median AMH levels were 26.61 pmol/L for those aged 20-25 years, 20.89 pmol/L for 26-30 years, 19.92 pmol/L for 31-35 years, 13.71 pmol/L for 36-40 years, 9.24 pmol/L for 41-45 years, and 0.68 pmol/L for 46-50 years. The recommended 2.5th to 97.5th percentiles of AMH level, as reference ranges for various age groups, were found to be: 10.63-55.64 pmol/L (20-25 years), 3.74-61.88 pmol/L (26-30 years), 5.49-47.56 pmol/L (31-35 years), 2.15-48.91 pmol/L (36-40 years), 0.92-41.26 pmol/L (41-45 years), and 0.14-5.10 pmol/L (46-50 years). Conclusions: This study (the first in Oman) determined the age-specific reference ranges of serum AMH in healthy Omani women in the age range of 20-50 years.

Seasonal variation and sleep patterns in a hot climate Arab Region.

PURPOSE: To describe the effect of seasonal variations on sleep patterns in a hot climate Arab region. METHODS: This is a cross-sectional study that included healthy Omani subjects of both genders between ages 18 and 59 years. Data for sleep pattern identification in summer and winter were collected from participants using an actigraphy wristband. RESULTS: Among 321 participants, in summer seasons, a polyphasic sleep pattern (40%) prevailed over other sleep patterns (P < 0.001). While in the winter season, monophasic sleep (31%) was the dominant pattern (P < 0.001). Subjects slept longer during the winter seasons with total hours of sleep during the day 48 min longer than in the summer, though the difference was not statistically significant (P > 0.05), while siesta duration in the summer was significantly longer (13 min, P < 0.01). In summer, the sleep quality was good (PSQI ≤ 5); however, it was poor (PSQI > 5) in winter (P < 0.05). Night sleep duration, daytime sleepiness, and sleep latency were not statistically different between the summer and winter seasons. CONCLUSION: Sleep patterns may be influenced by seasonal changes. A polyphasic sleep pattern prevailed in summer while a monophasic pattern was the predominant sleep pattern in winter. In summer, the sleep quality was good and the siesta duration was longer compared to the winter.

Gender-specific Reference Range for Serum Leptin in Omani Population.

Objectives: Leptin is a hormone that contributes to glucose homeostasis and food intake regulation via its action on the hypothalamus. Leptin level increases with obesity and overfeeding and decreases with energy deficiency. Serum leptin levels vary between different ethnic groups with no reports of its reference range in the Arabic population. We sought to determine gender-specific reference ranges for serum leptin in a cohort of the Arabic population and identify the cut-off value for different metabolic derangements. Methods: The study data were obtained from the records of 1198 subjects included in the Oman Family Study. The percentile method was used in the estimation reference range and the receiver operating characteristic to identify cut-off points for multiple metabolic derangements. Results: The reference range of serum leptin was 0.5-90.6 ng/mL, and it was not correlated with the age of the subjects. Higher leptin was observed in females compared to males (p < 0.001), and the reference range for serum leptin in females was 4.9-96.3 ng/mL compared to 0.25-48.8 ng/mL in males. The optimum cut-off value for leptin ranged between 24.1-28.9 ng/mL for metabolic syndrome, obesity, central obesity, and type 2 diabetes. Conclusions: We identified gender-specific reference ranges for serum leptin in a large cohort of Arabs. The optimum cut-off value for serum leptin to determine metabolic derangement with the highest sensitivity and specificity was 24.1-28.9 ng/mL. Future studies are needed to study the relative risk of higher serum leptin using prospective studies.

Biochemical, Hematological, and Immunological Biomarkers as Predictors for Intensive Care Unit Admission in Patients with COVID-19.

Objectives: To identify the biochemical, hematological, and immune biomarkers in COVID-19 patients on admission that are predictive of eventual admission to the intensive care unit (ICU). Methods: This retrospective cohort study was conducted on all confirmed COVID-19 cases hospitalized at Royal Hospital, Oman from 24 February to 30 July 2020. The demographic, clinical, and laboratory data were collected from the hospital information system. Patients were divided into two groups: non-ICU admitted group and ICU admitted group. Results: Out of 445 patients, 276 (62.0%) were male and 169 (38.0%) were female; 259 (58.2%) patients were admitted to COVID-19 general wards whereas 186 (41.8%) were admitted to ICU. Admission to ICU was more likely when patient had the following comorbidities: diabetes (OR = 1.8; 95% CI: 1.3-2.7), liver diseases (OR = 2.1; 95% CI: 1.1-4.3), and respiratory diseases (OR = 2.0; 95% CI: 1.1-3.7). Between ICU and non-ICU patients, there were significant differences in on-admission laboratory blood/serum parameters: total white blood cells (WBCs) count, lymphocytes count, C-reactive protein (CRP), ferritin, corrected calcium, interleukin 6 (IL-6), D-dimer, alanine transaminase (ALT), lactate dehydrogenase (LDH), albumin, and troponin. Conclusions: The current study identified the presence of the comorbidities (i.e., diabetes, liver diseases, and respiratory diseases) and on-admission laboratory blood and serum test results (i.e., WBC, lymphocytes, CRP, ferritin, corrected calcium, IL-6, D-dimer, ALT, LDH, albumin, and troponin) that are associated with ICU admission.

The diagnostic yield, candidate genes, and pitfalls for a genetic study of intellectual disability in 118 middle eastern families.

Global Developmental Delay/Intellectual disability (ID) is the term used to describe various disorders caused by abnormal brain development and characterized by impairments in cognition, communication, behavior, or motor skills. In the past few years, whole-exome sequencing (WES) has been proven to be a powerful, robust, and scalable approach for candidate gene discoveries in consanguineous populations. In this study, we recruited 215 patients affected with ID from 118 Middle Eastern families. Whole-exome sequencing was completed for 188 individuals. The average age at which WES was completed was 8.5 years. Pathogenic or likely pathogenic variants were detected in 32/118 families (27%). Variants of uncertain significance were seen in 33/118 families (28%). The candidate genes with a possible association with ID were detected in 32/118 (27%) with a total number of 64 affected individuals. These genes are novel, were previously reported in a single family, or cause strikingly different phenotypes with a different mode of inheritance. These genes included: AATK, AP1G2, CAMSAP1, CCDC9B, CNTROB, DNAH14, DNAJB4, DRG1, DTNBP1, EDRF1, EEF1D, EXOC8, EXOSC4, FARSB, FBXO22, FILIP1, INPP4A, P2RX7, PRDM13, PTRHD1, SCN10A, SCYL2, SMG8, SUPV3L1, TACC2, THUMPD1, XPR1, ZFYVE28. During the 5 years of the study and through gene matching databases, several of these genes have now been confirmed as causative of ID. In conclusion, understanding the causes of ID will help understand biological mechanisms, provide precise counseling for affected families, and aid in primary prevention.

Association of elevated glycated hemoglobin and obesity with afternoon napping for more than 1 h in young and middle-aged healthy adults.

Introduction: Sleep has different patterns followed worldwide and can be influenced by social, cultural, and environmental factors. Daytime napping is commonly practiced in different parts of the world with controversial results of its effect on glucose metabolism. The current study aims to examine the association of afternoon napping and night sleep duration with metabolic derangements. Methods: This is a cross-sectional study involving young adults and middle-aged subjects. Anthropometric measurements were taken for height and weight and hip and waist ratio. Consented subjects were asked to wear actigraphy for 1 week and run their usual daily activities. Home sleep apnea testing was performed to exclude obstructive sleep apnea. Subjects had been asked to come fasting on day seven for blood collection to test for fasting glucose, glycated hemoglobin, lipid profile, and insulin. Results: A total of 405 subjects were involved to complete the study (52% male, 48% female). The mean age of participants was 32.8 ± 11.5 years. The study indicated that the duration of afternoon napping was significantly associated with abnormal glycated hemoglobin (HbA1c > 5.7%) (p = 0.01) and body mass index (p = 0.046) independent of age, gender, and nocturnal sleep duration. Nocturnal sleep duration was associated with increased insulin level (p = 0.04). Conclusion: Afternoon napping is associated with an increased level of glycated hemoglobin and obesity and that may predispose to the development of type 2 diabetes mellitus.

Coexistence of sickle cell disease and systemic lupus erythematosus is associated with quantitative and qualitative impairments in circulating regulatory B cells.

The incidence of connective tissue diseases such as systemic lupus erythematous (SLE), in adult patients with sickle cell disease (SCD), appears to be increasing. The exact causes underlying this increased risk are still unknown, but a link with B regulatory (Breg) cells is possible as these cells suppress inflammatory responses, and maintain tolerance. Quantitative and qualitative analyses of circulating Breg cells were performed in a cohort of SCD patients with SLE, and their levels were correlated with key soluble mediators promoting autoreactive B cells. We demonstrated that levels of Breg cells were significantly decreased in SCD patients with SLE compared to patients with SCD only or healthy controls. Functional analysis of Breg cells from SCD patients with SLE revealed impairments in IL-10 production that correlated with lower levels of STAT3 phosphorylation, without abnormal expression of IL-10 receptor on Breg cells. On the other hand, BAFF levels were substantially elevated in SCD patients with SLE, but not significantly associated with Breg cell levels. Collectively, these results indicated numerical and functional deficits of Breg cells in SCD patients with SLE and their capacity to maintain tolerance and control inflammation is imbalanced, which leads to the development of autoimmune responses.

The Parental and Children Report of the Prevalence of Depressive Symptoms in Children and Adolescents Amid the COVID-19 Pandemic: A Cross-Sectional Study From Oman.

Objective: Studies from the past decades have shown that mood disorders are common during childhood and adolescence. This study aimed to estimate the point prevalence of depression in Omani children and adolescents during social distancing and lockdown and identify the risk factors for developing depressive symptoms during the COVID-19 pandemic. Methods: This is an analytical cross-sectional study conducted in May 2020, in which all young Omani people attending a mainstream school aged 8-18 years old were eligible to participate. Parents were asked to complete the online survey, which consisted of the parent version of the Mood and Feelings Questionnaire (MFQ-Parent). In addition, the option of a self-reported version (MFQ-Self) was provided in cases where children preferred to fill out the survey themselves. Logistic regression was used to identify the contributing socio-demographic variables associated with depressive symptoms. Results: A total of 445 participants completed the MFQ, out of which 72.1% were parents, and 27.9% were children, adolescents and young people. 13.9% of children and adolescents exhibited depressive symptoms during the COVID-19 pandemic in Oman. The presence of depressive symptoms was associated with increased food intake (OR 1.81, 95% CI 1.00-3.29, p-value <0.05), longer use of smartphones (OR 2.72, 95% CI 1.56-4.73, p-value <0.001), whereas additional entertainment activities during lockdown were protective against depression (OR 0.35 95% CI 0.19-0.64, p-value <0.001). Conclusion: This study from Oman concurs with recent reports of depression being common during the COVID-19 pandemic. Concerted efforts are needed to mitigate this trend and identify high-risk groups during the lockdown period.

Short and long-term immune changes in different severity groups of COVID-19 disease.

BACKGROUND: There are limited data on short- versus long-term changes in adaptive immune response across different COVID-19 disease severity groups. METHODS: A multicenter prospective study of 140 adult patients with COVID-19 (a total of 325 samples) were analyzed for inflammatory markers and lymphocyte subsets at presentation, week 2, and week 24. RESULTS: Inflammatory markers at presentation were higher in the critical/severe than in moderate and mild groups. A predominance of memory B cell response in the mild and moderate group was noted by week 2. In contrast, the immune system in the severe/critical group was dysfunctional, with expansion of exhausted CD8+ T cells and atypical memory B cells. By 24 weeks, there was a possible trend of normalization. CONCLUSION: There was substantial difference in the degree of inflammation and distribution of different B and T cell subsets in the different disease severity groups. Despite the initial dysfunctional immune response in the severe/critical group, a comparable memory B and CD8+ T cell responses to the mild group was achieved at 24 weeks.

The Genetic Spectrum of Familial Hypertriglyceridemia in Oman.

Familial hypertriglyceridemia (F-HTG) is an autosomal disorder that causes severe elevation of serum triglyceride levels. It is caused by genetic alterations in LPL, APOC2, APOA5, LMF1, and GPIHBP1 genes. The mutation spectrum of F-HTG in Arabic populations is limited. Here, we report the genetic spectrum of six families of F-HTG of Arab ancestry in Oman. Methods: six Omani families affected with triglyceride levels >11.2 mmol/L were included in this study. Ampli-Seq sequencing of the selected gene panels was performed. Whole-exome sequencing and copy number variant analysis were also performed in cases with negative exome results. Three novel pathogenic missense variants in the LPL gene were identified, p.M328T, p.H229L, and p.S286G, along with a novel splice variant c.1322+15T > G. The LPL p.H229L variant existed in double heterozygous mutation with the APOA5 gene p.V153M variant. One family had a homozygous mutation in the LMF1 gene (c.G107A; p.G36D) and a heterozygous mutation in the LPL gene (c.G106A; p.D36N). All affected subjects did not have a serum deficiency of LPL protein. Genetic analysis in one family did not show any pathogenic variants even after whole-exome sequencing. These novel LPL and APOA5 mutations are not reported in other ethnic groups. This suggests that patients with F-HTG in Oman have a founder effect and are genetically unique. This warrants further analysis of patients of F-HTG in the Middle East for preventative and counseling purposes to limit the spread of the disease in a population of high consanguinity.

Socio-Demographic and Clinical Profiles of Adult Attention Deficit Hyperactivity Disorder Patients in a University Hospital in Oman.

Objectives: Although attention deficit hyperactivity disorder (ADHD) is typically considered a condition affecting children, there is evidence that children diagnosed with ADHD continue to suffer from this condition after the age of 18. This study aimed to describe the socio-demographic and clinical characteristics of adult ADHD patients in Oman and evaluate their association with the disorder's different subtypes. Methods: This retrospective study included adult patients with ADHD from the outpatient clinic at Sultan Qaboos University Hospital, Muscat, Oman. Data from medical records from January 2018 to April 2020 were collected. Socio-demographic characteristics, clinical profiles and psychiatric comorbidities were examined. Results: This study included 100 adults who fulfilled the standard diagnosis of ADHD, with 54.0% (n = 54) and 46.0% (n = 46) from the inattentive and combined subtypes, respectively. It was found that ADHD was more prevalent among males (64.0%) than females (36.0%), with the inattentive subtype being more predominant among females. The ADHD patients with the inattentive subtype were associated with comorbid substance use disorders (odds ratio [OR] = 11.29; P = 0.049), personality disorders (OR = 7.96; P = 0.017) and major depressive disorder (OR = 15.94; P = 0.002) compared to patients predominantly with the combined subtype. Conclusion: This study echoes the findings from the current literature that adult patients with ADHD commonly have comorbid psychiatric disorders, leading to significant functional impairment. Psychiatric comorbidities must be identified and urgently treated for better clinical and functional outcomes in adult patients with ADHD.

Single Gene Mutations in Pkd1 or Tsc2 Alter Extracellular Vesicle Production and Trafficking.

Patients with autosomal dominant polycystic kidney disease (ADPKD) and tuberous sclerosis complex (TSC) are born with normal or near-normal kidneys that later develop cysts and prematurely lose function. Both renal cystic diseases appear to be mediated, at least in part, by disease-promoting extracellular vesicles (EVs) that induce genetically intact cells to participate in the renal disease process. We used centrifugation and size exclusion chromatography to isolate the EVs for study. We characterized the EVs using tunable resistive pulse sensing, dynamic light scattering, transmission electron microscopy, and Western blot analysis. We performed EV trafficking studies using a dye approach in both tissue culture and in vivo studies. We have previously reported that loss of the Tsc2 gene significantly increased EV production and here demonstrate that the loss of the Pkd1 gene also significantly increases EV production. Using a cell culture system, we also show that loss of either the Tsc2 or Pkd1 gene results in EVs that exhibit an enhanced uptake by renal epithelial cells and a prolonged half-life. Loss of the primary cilia significantly reduces EV production in renal collecting duct cells. Cells that have a disrupted Pkd1 gene produce EVs that have altered kinetics and a prolonged half-life, possibly impacting the duration of the EV cargo effect on the recipient cell. These results demonstrate the interplay between primary cilia and EVs and support a role for EVs in polycystic kidney disease pathogenesis.

Mucolipidosis Type IV in Omani Families with a Novel MCOLN1 Mutation: Search for Evidence of Founder Effect.

Mucolipidosis Type IV (MLIV) is caused by a deficiency of the mucolipin cation channel encoded by Mucolipin TRP Cation Channel 1 gene (MCOLN1). It is a slowly progressive neurodevelopmental and neurodegenerative disorder causing severe psychomotor developmental delay and progressive visual impairment, which is often misdiagnosed as cerebral palsy. We describe six patients with MLIV from two Omani families with a novel c.237+5G>A mutation in the MCOLN1 gene predicted to affect mRNA splicing. Mutation screening with a high-resolution melting (HRM) assay in a large population sample did not detect this mutation in control subjects. This report highlights the importance of considering MLIV in the differential diagnosis of patients in a pediatric age group with cerebral palsy-like presentation. Although the same rare mutation was seen in two apparently unrelated families, this was not seen in the sample screened from the general population. The HRM assay provides a cost-effective assay for population screening for the c.237+5G>A mutation.

Tsc2 mutation induces renal tubular cell nonautonomous disease.

TSC renal cystic disease is poorly understood and has no approved treatment. In a new principal cell-targeted murine model of Tsc cystic disease, the renal cystic epithelium is mostly composed of type A intercalated cells with an intact Tsc2 gene confirmed by sequencing, although these cells exhibit a Tsc-mutant disease phenotype. We used a newly derived targeted murine model in lineage tracing and extracellular vesicle (EV) characterization experiments and a cell culture model in EV characterization and cellular induction experiments to understand TSC cystogenesis. Using lineage tracing experiments, we found principal cells undergo clonal expansion but contribute very few cells to the cyst. We determined that cystic kidneys contain more interstitial EVs than noncystic kidneys, excrete fewer EVs in urine, and contain EVs in cyst fluid. Moreover, the loss of Tsc2 gene in EV-producing cells greatly changes the effect of EVs on renal tubular epithelium, such that the epithelium develops increased secretory and proliferative pathway activity. We demonstate that the mTORC1 pathway activity is independent form the EV production, and that the EV effects for a single cell line can vary significantly. TSC cystogenesis involves significant contribution from genetically intact cells conscripted to the mutant phenotype by mutant cell derived EVs.

Electronic nicotine delivery systems exhibit reduced bronchial epithelial cells toxicity compared to cigarette: the Replica Project.

Electronic nicotine delivery systems (ENDS) may reduce health risks associated with chronic exposure to smoke and their potential benefits have been the matter of intense scientific debate. We aimed to replicate three published studies on cytotoxic and inflammatory effects of cigarette smoke and ENDS aerosol in an independent multi-center ring study. We aimed to establish the reliability of results and the robustness of conclusions by replicating the authors' experimental protocols and further validating them with different techniques. Human bronchial epithelial cells (NCI-H292) were exposed to cigarette whole smoke and vapor phase and to aerosol from ENDS. We also assessed the inflammatory cytokines interleukin-6 and interleukin-8 and the remodeling mediator matrix metalloproteinase-1. We replicated cell viability results and confirmed that almost 80% of cytotoxic effects are due to volatile compounds in the vapor phase of smoke. Our findings substantiated the reduced cytotoxic effects of ENDS aerosol. However, our data on inflammatory and remodeling activity triggered by smoke differed significantly from those in the original reports. Taken together, independent data from multiple laboratories clearly demonstrated the reduced toxicity of ENDS products compared to cigarettes.