Synthetic abnormal mast cell particles successfully mimic neoplastic mast cells by flow cytometry.

Clinical flow cytometry laboratories require quality control materials for assay development, validation, and performance monitoring, including new reagent lot qualification. However, finding suitable controls for populations with uncommonly expressed antigens or for rare populations, such as mast cells, can be difficult. To that end, we evaluated synthetic abnormal mast cell particles (SAMCP), developed together with, and manufactured by, Slingshot Biosciences. The SAMCP's were designed to phenotypically mimic abnormal neoplastic mast cells: they were customized to have the same light scatter and autofluorescence properties of mast cells, along with surface antigen levels of CD45, CD33, CD117, CD2, CD25, and CD30 consistent with that seen in mast cell disease. We evaluated several performance characteristics of these particles using ARUP's high sensitivity clinical mast cell assay, including limit of detection, off-target activity and FMO controls, precision, scatter properties of the particles utilizing several different cytometer platforms, and particle antigen stability. The phenotype of the SAMCP mimicked abnormal mast cells, and they could be distinguished from normal native mast cells. FMO controls demonstrated specificity of each of the markers, and no off-target binding was detected. The limit of detection of the particles spiked into normal bone marrow was found to be ≤0.003% in a limiting dilution assay. The mast cell particles were found to perform similarly on Becton Dickinson Lyric, Cytek Aurora, and Beckman Coulter Navios and CytoFLEX platforms. Within run and between run precision were less than 10% CV. SAMCP were stable up to 13 days with minimal loss of antigen fluorescence intensity. The SAMCP's were able to successfully mimic neoplastic mast cells based on the results of our high sensitivity mast cell flow cytometry panel. These synthetic cell particles represent an exciting and innovative technology, which can fulfill vital needs in clinical flow cytometry such as serving as standardized control materials for assay development and performance monitoring.

Combining physical & cognitive training with iPSC-derived dopaminergic neuron transplantation promotes graft integration & better functional outcome in parkinsonian marmosets.

Parkinson's disease (PD) is a relentlessly progressive and currently incurable neurodegenerative disease with significant unmet medical needs. Since PD stems from the degeneration of midbrain dopaminergic (DA) neurons in a defined brain location, PD patients are considered optimal candidates for cell replacement therapy. Clinical trials for cell transplantation in PD are beginning to re-emerge worldwide with a new focus on induced pluripotent stem cells (iPSCs) as a source of DA neurons since they can be derived from adult somatic cells and produced in large quantities under current good manufacturing practices. However, for this therapeutic strategy to be realized as a viable clinical option, fundamental translational challenges need to be addressed including the manufacturing process, purity and efficacy of the cells, the method of delivery, the extent of host reinnervation and the impact of patient-centered adjunctive interventions. In this study we report on the impact of physical and cognitive training (PCT) on functional recovery in the nonhuman primate (NHP) model of PD after cell transplantation. We observed that at 6 months post-transplant, the PCT group returned to normal baseline in their daily activity measured by actigraphy, significantly improved in their sensorimotor and cognitive tasks, and showed enhanced synapse formation between grafted cells and host cells. We also describe a robust, simple, efficient, scalable, and cost-effective manufacturing process of engraftable DA neurons derived from iPSCs. This study suggests that integrating PCT with cell transplantation therapy could promote optimal graft functional integration and better outcome for patients with PD.

A Pharmacokinetic and Analgesic Efficacy Study of Carprofen in Female CD1 Mice.

The minimization of pain in research animals is a scientific and ethical necessity. Carprofen is commonly used for pain management in mice; however, some data suggest that the standard dosage of 5 mg/kg may not provide adequate analgesia after surgery. We hypothesized that a higher dose of carprofen in mice would reduce pain-associated behaviors and improve analgesia without toxic effects. A pharmacokinetic study was performed in mice given carprofen subcutaneously at 10 or 20 mg/kg. Plasma concentrations were measured at 0.25, 0.5, 1, 2, 4, 8, 12, 24, and 48 h after dosing (n = 3 per time point and treatment). At these doses, plasma levels were above the purported therapeutic level for at least 12 and 24 h, respectively, with respective half-lives of 14.9 and 10.2 h. For the efficacy study, 10 mice per group received anesthesia with or without an ovariectomy. Mice were then given 5 or 10 mg/kg of carprofen, or saline subcutaneously every 12 h. Orbital tightening, arched posture, wound licking, rearing, grooming, nesting behavior, and activity were assessed before surgery and at 4, 8, 12, 24, and 48 h after surgery. The von Frey responses were assessed before and at 4, 12, 24, and 48 h after surgery. The efficacy study showed that all surgery groups had significantly higher scores for orbital tightening, arched posture, and wound licking than did the anesthesia-only groups at 4, 8, 12, and 24-h time points. At the 8 h time point, the surgery groups treated with carprofen had significantly lower arched posture scores than did the surgery group treated with saline only. No significant differences were found between carprofen treatment groups for rearing, grooming, von Frey, activity, or nesting behavior at any time point. These results indicate that subcutaneous carprofen administered at these doses does not provide sufficient analgesia to alleviate postoperative pain in female CD1 mice.

Nanobodies: Robust miniprotein binders in biomedicine.

Variable domains of heavy chain-only antibodies (VHH), also known as nanobodies (Nbs), are monomeric antigen-binding domains derived from the camelid heavy chain-only antibodies. Nbs are characterized by small size, high target selectivity, and marked solubility and stability, which collectively facilitate high-quality drug development. In addition, Nbs are readily expressed from various expression systems, including E. coli and yeast cells. For these reasons, Nbs have emerged as preferred antibody fragments for protein engineering, disease diagnosis, and treatment. To date, two Nb-based therapies have been approved by the U.S. Food and Drug Administration (FDA). Numerous candidates spanning a wide spectrum of diseases such as cancer, immune disorders, infectious diseases, and neurodegenerative disorders are under preclinical and clinical investigation. Here, we discuss the structural features of Nbs that allow for specific, versatile, and strong target binding. We also summarize emerging technologies for identification, structural analysis, and humanization of Nbs. Our main focus is to review recent advances in using Nbs as a modular scaffold to facilitate the engineering of multivalent polymers for cutting-edge applications. Finally, we discuss remaining challenges for Nb development and envision new opportunities in Nb-based research.

High-dose Meloxicam Provides Improved Analgesia in Female CD1 Mice: A Pharmacokinetic and Efficacy Study.

Meloxicam is a nonsteroidal anti-inflammatory analgesic drug that is often used in mice. However, doses of 1 to 5 mg/kg given twice daily were recently reported to provide inadequate analgesia. Some studies suggest that doses of up to 20 mg/kg may be necessary for adequate pain management. We investigated the analgesia provided by a high-dose of meloxicam in female CD1 mice. Pharmacokinetic analyses demonstrated that a subcutaneous injection of 10 mg/kg or 20 mg/kg of meloxicam produced therapeutic plasma concentrations for at least 12 h. Ovariectomies via ventral laparotomy were performed to assess analgesic efficacy. Mice were treated immediately before surgery with a high-dose of 10 mg/kg, a low-dose of 2.5 mg/kg, or saline, followed by every 12 h for 36 h. At 3, 6, 12, 24, and 48 h after surgery, mice were assessed for pain based on the following behaviors: distance traveled, time mobile, grooming, rearing, hunched posture, orbital tightening, and von Frey. Initially, some mice received a 20-mg/kg loading dose followed by 10 mg/kg every 12 h. This regimen caused severe morbidity and mortality in 2 mice. Subsequently, this regimen was abandoned, and mice assigned to the high-dose group received 10 mg/kg every 12 h. Mice that received the 10-mg/kg dose after surgery showed less orbital tightening between 3 to 6 h and reduced frequency of hunched posture for 48 h compared with mice that received either the low-dose or saline. However, mice were significantly less mobile for 6 to 12 h after surgery regardless of treatment. These data indicate that a meloxicam dose of 10 mg/kg every 12 h provides better analgesia than a 2.5-mg/kg dose but does not completely alleviate pain.

Effects of Helicobacter pylori therapy on gut microbiota: a systematic review and meta-analysis.

BACKGROUND: Although indications for evaluation and treatment of Helicobacter pylori (H. pylori) infection are broadening to include primary prevention for gastric adenocarcinoma, potential adverse effects on gut microbiota have been raised. We performed a systematic review and meta-analysis to evaluate the effects of H. pylori therapy on gut microbiota. METHODS: PubMed, EMBASE, Cochrane Library and Web of Science (to 4/2021) were searched for studies quantitatively evaluating microbiota before and after H. pylori therapy. Meta-analysis was performed to assess early (<1 year) and long-term (≥1 year) effects on gut microbiota after H. pylori treatment. Subgroup analysis evaluating the effects of H. pylori therapy with addition of probiotics on gut microbiota was also performed. RESULTS: Thirty studies (N=1,218) met the criteria. Early after H. pylori therapy, intestinal microbial diversity was reduced in nearly all studies. At the genus level, reduction in the abundance of Enterococcus, while increase in Lactobacillus, Bifidobacterium, and Bacteroides counts were observed. However, Lactobacillus, Bifidobacterium, Bacteroides, and Enterococcus counts remained stable in patients who received probiotics with H. pylori therapy. At the phylum level, the relative abundance of Actinobacteria and Firmicutes increased after treatment. At ≥1 year, intestinal microbial diversity normalized in six of seven studies. No differences in the relative abundance of Actinobacteria, Firmicute, Bacteroidetes, and Proteobacteria were observed ≥1 year after therapy. CONCLUSION: The impact of H. pylori therapy on gut microbiota appears transient with early changes largely resolving after one year. Probiotics may reduce the early impact of H. pylori therapy on gut microbiota.

Distribution and frequency of tyrosine kinase inhibitor-associated long-term complications in children with chronic myeloid leukemia.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) improve outcomes for pediatric malignancies characterized by specific gene rearrangements and mutations; however, little is known about the long-term impact of TKI exposure. Our objective was to assess the incidence and type of late-onset TKI-related toxicities in children with chronic myeloid leukemia (CML). METHODS: We reviewed medical records from patients diagnosed with CML between 2006 and 2019 at <21 years of age and prescribed one or more TKIs. Patients treated with stem cell transplant were excluded. Outcomes were captured beginning at 1 year after CML diagnosis. Outcome incidence was described overall and stratified by TKI exposure during the data-capture period. RESULTS: Twenty-two eligible TKI-exposed patients with CML were identified. The median follow-up was 6.0 years (range: 2.2-14.3). All pericardial (n = 3) or pleural (n = 3) effusion outcomes occurred in patients treated with TKIs during the data-capture period. Other outcomes included hypertension (n = 2), ectopy on electrocardiogram (n = 2), and gastrointestinal bleed (n = 1). All outcomes were graded as mild to moderate: some resulted in a temporary discontinuation of TKI, but none led to a change in TKI. No differences were noted in outcome incidence by type of TKI exposure. CONCLUSIONS: TKIs have substantially improved prognosis for subsets of childhood leukemia, but there are limited long-term data to inform exposure-based risk for late-onset complications and screening. Our results suggest that TKI-exposed survivors may be at risk for long-term outcomes that extend well into survivorship.

Lung Cancer: Diagnosis, Treatment Principles, and Screening.

Lung cancer is the second most common cancer in men and women in the United States; however, it remains the leading cause of cancer-related death in the United States and worldwide. The most common but nonspecific symptom of lung cancer is cough. Associated symptoms, including hemoptysis or shortness of breath, or systemic symptoms, including anorexia or weight loss, greatly increase the likelihood of having lung cancer. Referral to a multidisciplinary lung cancer team, imaging, and confirmation through sputum cytology, thoracentesis, fine-needle aspiration, or mediastinoscopy are recommended. If lung cancer is confirmed, treatment options vary based on staging, histology, immunotherapy biomarker testing, and patient health status. Treatments include surgical resection, immunotherapy, chemotherapy, and/or radiotherapy. Family physicians should focus on primary prevention of lung cancer by encouraging tobacco cessation and early recognition by screening at-risk individuals and following guidelines for pulmonary nodules. As of 2021, the U.S. Preventive Services Task Force recommends annual lung cancer screening using low-dose computed tomography starting at 50 years of age in patients with a 20 pack-year history.

Sotalol versus amiodarone for postoperative junctional tachycardia after congenital heart surgery.

BACKGROUND: Junctional ectopic tachycardia (JET) is a common arrhythmia after congenital heart disease surgery. There is variability in the choice of antiarrhythmic therapy, with amiodarone used commonly. Intravenous (IV) sotalol is a newly available agent that may be useful for JET. OBJECTIVE: The purpose of this study was to evaluate the safety and efficacy of IV sotalol for postoperative JET and compare outcomes with IV amiodarone. METHODS: This is a retrospective single-center study of all patients who received IV sotalol or IV amiodarone for postoperative JET at Texas Children's Hospital from December 15, 2015, to December 15, 2020. Data included antiarrhythmic efficacy, hemodynamics, and adverse effects. Successful JET control was defined as a decrease in JET rate to <170 beats/min (or decrease by >20%), or conversion to sinus rhythm, with persistent control over 24 hours without requiring alternative antiarrhythmics or mechanical support. RESULTS: A total of 32 patients (median age 71 days; interquartile range 17-221 days) received IV amiodarone (n = 20 [62%]) or IV sotalol (n = 12 [38%]) for postoperative JET. Amiodarone was successful in treating JET in 75% of cases; sotalol was successful in 83%. The JET rate decreased faster over the first 90 minutes after a sotalol bolus (25 beats/min per hour) than after an amiodarone bolus (8 beats/min per hour) (P < .01); no heart rate difference was seen after 24 hours. Amiodarone infusion was discontinued early because of hypotension/bradycardia in 2 patients; this was not required in any patients receiving sotalol. CONCLUSION: For children with postoperative JET, both IV sotalol and amiodarone are safe and efficacious. IV sotalol may lead to a faster improvement in heart rate.

Increased PD-L1 and p16 expression are common in oropharyngeal squamous cell carcinoma.

Overexpression of p16 is closely related to human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (SCC) and pertains a prognostic relevance. Programmed cell death 1-ligand 1 (PD-L1) is another important marker, as anti-PD-L1 immunotherapy is available. Retrospective analysis of 57 cases of the SCC involving oropharynx (27 cases), hypopharynx (5 cases), larynx (11 cases), and oral cavity (14 cases) was performed. Each case was scrutinized for the basaloid morphology, p16, and PD-L1 expression. Basaloid morphology was identified in 47% of total cases. The majority of basaloid SCC variants were located in the oropharynx (89%). High expression of p16 was mostly observed in the oropharynx. High PD-L1 expression was seen predominantly in oropharyngeal and hypopharyngeal locations. Further studies in a larger cohort are necessary to correlate PD-L1 and p16 expression with survival.

Iron-Storage Disorder Presenting as Chronic Diarrhea.

The involvement of the endocrine pancreas leading to bronze diabetes is well studied. However, little is known about the pathophysiology of iron dysregulation involving the exocrine pancreas. We present a unique association between the exocrine pancreas and iron dysregulation. A 45-year-old female presented with chronic diarrhea and low fecal elastase indicative of pancreatic exocrine dysfunction. MRI of the abdomen/pelvis showed iron deposition in the pancreas, suggesting an associated iron-storage disorder without features suggesting chronic pancreatitis. Association of an iron-storage disorder with pancreatic exocrine dysfunction has been reported only in one other case report. Pancreatic exocrine dysfunction can be directly associated with an iron-storage disorder that involves the pancreas. This should be included in the differential and diagnostic work-up of chronic diarrhea of unclear etiology. Based on the literature, we have highlighted the potential pathophysiology relevant to the case.

ATP1A3-Encoded Sodium-Potassium ATPase Subunit Alpha 3 D801N Variant Is Associated With Shortened QT Interval and Predisposition to Ventricular Fibrillation Preceded by Bradycardia.

Background Pathogenic variation in the ATP1A3-encoded sodium-potassium ATPase, ATP1A3, is responsible for alternating hemiplegia of childhood (AHC). Although these patients experience a high rate of sudden unexpected death in epilepsy, the pathophysiologic basis for this risk remains unknown. The objective was to determine the role of ATP1A3 genetic variants on cardiac outcomes as determined by QT and corrected QT (QTc) measurements. Methods and Results We analyzed 12-lead ECG recordings from 62 patients (male subjects=31, female subjects=31) referred for AHC evaluation. Patients were grouped according to AHC presentation (typical versus atypical), ATP1A3 variant status (positive versus negative), and ATP1A3 variant (D801N versus other variants). Manual remeasurements of QT intervals and QTc calculations were performed by 2 pediatric electrophysiologists. QTc measurements were significantly shorter in patients with positive ATP1A3 variant status (P<0.001) than in patients with genotype-negative status, and significantly shorter in patients with the ATP1A3-D801N variant than patients with other variants (P<0.001). The mean QTc for ATP1A3-D801N was 344.9 milliseconds, which varied little with age, and remained <370 milliseconds throughout adulthood. ATP1A3 genotype status was significantly associated with shortened QTc by multivariant regression analysis. Two patients with the ATP1A3-D801N variant experienced ventricular fibrillation, resulting in death in 1 patient. Rare variants in ATP1A3 were identified in a large cohort of genotype-negative patients referred for arrhythmia and sudden unexplained death. Conclusions Patients with AHC who carry the ATP1A3-D801N variant have significantly shorter QTc intervals and an increased likelihood of experiencing bradycardia associated with life-threatening arrhythmias. ATP1A3 variants may represent an independent cause of sudden unexplained death. Patients with AHC should be evaluated to identify risk of sudden death.

TBX5-encoded T-box transcription factor 5 variant T223M is associated with long QT syndrome and pediatric sudden cardiac death.

Long QT syndrome (LQTS) is a genetic disease resulting in a prolonged QT interval on a resting electrocardiogram, predisposing affected individuals to polymorphic ventricular tachycardia and sudden death. Although a number of genes have been implicated in this disease, nearly one in four individuals exhibiting the LQTS phenotype are genotype-negative. Whole-exome sequencing identified a missense T223M variant in TBX5 that cosegregates with prolonged QT interval in a family with otherwise genotype-negative LQTS and sudden death. The TBX5-T223M variant was absent among large ostensibly healthy populations (gnomAD) and predicted to be pathogenic by in silico modeling based on Panther, PolyPhen-2, Provean, SIFT, SNAP2, and PredictSNP prediction tools. The variant was located in a highly conserved region of TBX5 predicted to be part of the DNA-binding interface. A luciferase assay identified a 57.5% reduction in the ability of TBX5-T223M to drive expression at the atrial natriuretic factor promotor compared to wildtype TBX5 in vitro. We conclude that the variant is pathogenic in this family, and we put TBX5 forward as a disease susceptibility allele for genotype-negative LQTS. The identification of this familial variant may serve as a basis for the identification of previously unknown mechanisms of LQTS with broader implications for cardiac electrophysiology.

Human Induced Pluripotent Stem Cell Derived Sensory Neurons are Sensitive to the Neurotoxic Effects of Paclitaxel.

Chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting adverse event associated with treatment with paclitaxel and other chemotherapeutic agents. The prevention and treatment of CIPN are limited by a lack of understanding of the molecular mechanisms underlying this toxicity. In the current study, a human induced pluripotent stem cell-derived sensory neuron (iPSC-SN) model was developed for the study of chemotherapy-induced neurotoxicity. The iPSC-SNs express proteins characteristic of nociceptor, mechanoreceptor, and proprioceptor sensory neurons and show Ca2+ influx in response to capsaicin, α,ß-meATP, and glutamate. The iPSC-SNs are relatively resistant to the cytotoxic effects of paclitaxel, with half-maximal inhibitory concentration (IC50 ) values of 38.1 µM (95% confidence interval (CI) 22.9-70.9 µM) for 48-hour exposure and 9.3 µM (95% CI 5.7-16.5 µM) for 72-hour treatment. Paclitaxel causes dose-dependent and time-dependent changes in neurite network complexity detected by ßIII-tubulin staining and high content imaging. The IC50 for paclitaxel reduction of neurite area was 1.4 µM (95% CI 0.3-16.9 µM) for 48-hour exposure and 0.6 µM (95% CI 0.09-9.9 µM) for 72-hour exposure. Decreased mitochondrial membrane potential, slower movement of mitochondria down the neurites, and changes in glutamate-induced neuronal excitability were also observed with paclitaxel exposure. The iPSC-SNs were also sensitive to docetaxel, vincristine, and bortezomib. Collectively, these data support the use of iPSC-SNs for detailed mechanistic investigations of genes and pathways implicated in chemotherapy-induced neurotoxicity and the identification of novel therapeutic approaches for its prevention and treatment.

Frequency and Outcomes of Elevated Perioperative Lactate Levels in Adult Congenital Heart Disease Patients Undergoing Cardiac Surgery.

OBJECTIVES: To assess whether lactate levels are associated with clinical outcomes in adult congenital heart disease patients who undergo cardiac surgery. DESIGN: Retrospective study. SETTING: Single quaternary academic referral center. PARTICIPANTS: Adult congenital heart disease patients (≥18 y old) with congenital heart disease undergoing cardiac surgery with cardiopulmonary bypass. INTERVENTIONS: Participants were classified into 3 groups according to their peak arterial lactate level within the first 48 hours of surgery. MEASUREMENTS AND MAIN RESULTS: In-hospital 30-day mortality, hospital and intensive care unit length of stay, duration of mechanical ventilation after surgery, acute kidney injury defined by Acute Kidney Injury Network criteria, and intensive care unit and hospital readmission within 30 days of surgery were examined. There was no significant difference among different lactate level groups in acute kidney injury, hospital length of stay, intensive care unit length of stay, hours of mechanical ventilation, need for redo surgery, or rates of hospital or intensive care unit readmission. In multivariable analysis, which included cardiopulmonary bypass time, redo surgery, nonelective case, and the adult congenital heart disease complexity score, lactate levels were not a significant predictor of either acute kidney injury or hospital length of stay. CONCLUSIONS: The appeal of using lactate levels to risk stratify-patients or to develop a model to predict mortality and morbidity has potential merit, but currently there is insufficient evidence to use lactate levels as a predictor of outcomes in adult patients with congenital heart disease undergoing cardiac surgery.

Incidentally identified genetic variants in arrhythmogenic right ventricular cardiomyopathy-associated genes among children undergoing exome sequencing reflect healthy population variation.

BACKGROUND: With expanding use of clinical whole exome sequencing (WES), genetic variants of uncertain significance are increasingly identified. As pathologic mutations in genes associated with arrhythmogenic right ventricular cardiomyopathy (ARVC) carry a risk of sudden death, determining the diagnostic relevance of incidentally identified variants associated with these genes is critical. METHODS: WES variants from a large, predominantly pediatric cohort (N = 7,066 probands) were obtained for nine ARVC-associated genes (Baylor Miraca). For comparison, a control cohort was derived from the gnomAD database and an ARVC case cohort (N = 1,379 probands) was established from ARVC cases in the literature. Topologic mapping was performed and signal-to-noise analysis was conducted normalizing WES, or case variants, against control variant frequencies. Retrospective chart review was performed of WES cases evaluated clinically (Texas Children's Hospital). RESULTS: Incidentally identified variants occurred in 14% of WES referrals and localized to genes which were rare among ARVC cases yet similar to controls. Amino acid-level signal-to-noise analysis of cases demonstrated "pathologic hotspots" localizing to critical domains of PKP2 and DSG2 while WES variants did not. PKP2 ARM7 and ARM8 domains and DSG2 N-terminal cadherin-repeat domains demonstrated high pathogenicity while normalized WES variant frequency was low. Review of clinical data available on WES referrals demonstrated none with evidence of ARVC among variant-positive individuals. CONCLUSIONS: Incidentally identified variants are common among pediatric WES testing with gene frequencies similar to "background" variants. Incidentally identified variants are unlikely to be pathologic.

Reference Transcriptome for Deriving Marmoset Induced Pluripotent Stem Cells.

Limited access to primary tissue from various nonhuman primate (NHP) species represents a significant unmet need that hampers progress in understanding unique cellular diversity and gene regulation of specific tissues and organs in stem cell translational research. Most comparative biology studies have been limited to using postmortem tissue usually frozen specimens with limited utility for research. The generation of induced pluripotent stem cell (iPSC) lines from somatic cells, such as adult skin or blood cells, offers an alternative to invasive and ethically controversial interventions for acquiring tissue. Pluripotent iPSCs have virtually an unlimited capacity to proliferate and differentiate into all cell types of the body. We are generating high-quality validated NHP iPSC lines to offer to scientific community and facilitate their research programs. We use the non-integrative episomal vector system to generate iPSCs from NHP skin biopsies. In this chapter we describe the validation of NHP iPSC lines by confirming pluripotency and their propensity to differentiate into all three germ layers ectoderm, mesoderm, and endoderm according to established standards and measurable limits for a set of marker genes incorporated into a scorecard.

Cariogenic potential of sweet flavors in electronic-cigarette liquids.

BACKGROUND: Most electronic-cigarette liquids contain propylene glycol, glycerin, nicotine and a wide variety of flavors of which many are sweet. Sweet flavors are classified as saccharides, esters, acids or aldehydes. This study investigates changes in cariogenic potential when tooth surfaces are exposed to e-cigarette aerosols generated from well-characterized reference e-liquids with sweet flavors. METHODS: Reference e-liquids were prepared by combining 20/80 propylene glycol/glycerin (by volume fraction), 10 mg/mL nicotine, and flavors. Aerosols were generated by a Universal Electronic-Cigarette Testing Device (49.2 W, 0.2 Ω). Streptococcus mutans (UA159) were exposed to aerosols on tooth enamel and the biological and physiochemical parameters were measured. RESULTS: E-cigarette aerosols produced four-fold increase in microbial adhesion to enamel. Exposure to flavored aerosols led to two-fold increase in biofilm formation and up to a 27% decrease in enamel hardness compared to unflavored controls. Esters (ethyl butyrate, hexyl acetate, and triacetin) in e-liquids were associated with consistent bacteria-initiated enamel demineralization, whereas sugar alcohol (ethyl maltol) inhibited S. mutans growth and adhesion. The viscosity of the e-liquid allowed S. mutans to adhere to pits and fissures. Aerosols contained five metals (mean ± standard deviation): calcium (0.409 ± 0.002) mg/L, copper (0.011 ± 0.001) mg/L, iron (0.0051 ± 0.0003) mg/L, magnesium (0.017 ± 0.002) mg/L, and silicon (0.166 ± 0.005) mg/L. CONCLUSIONS: This study systematically evaluated e-cigarette aerosols and found that the aerosols have similar physio-chemical properties as high-sucrose, gelatinous candies and acidic drinks. Our data suggest that the combination of the viscosity of e-liquids and some classes of chemicals in sweet flavors may increase the risk of cariogenic potential. Clinical investigation is warranted to confirm the data shown here.