Structure-activity relationship of dihydropyridines for rhabdomyosarcoma.

Childhood muscle-related cancer rhabdomyosarcoma is a rare disease with a 50-year unmet clinical need for the patients presented with advanced disease. The rarity of ∼350 cases per year in North America generally diminishes the viability of large-scale, pharmaceutical industry driven drug development efforts for rhabdomyosarcoma. In this study, we performed a large-scale screen of 640,000 compounds to identify the dihydropyridine (DHP) class of anti-hypertensives as a priority compound hit. A structure-activity relationship was uncovered with increasing cell growth inhibition as side chain length increases at the ortho and para positions of the parent DHP molecule. Growth inhibition was consistent across n = 21 rhabdomyosarcoma cell line models. Anti-tumor activity in vitro was paralleled by studies in vivo. The unexpected finding was that the action of DHPs appears to be other than on the DHP receptor (i.e., L-type voltage-gated calcium channel). These findings provide the basis of a medicinal chemistry program to develop dihydropyridine derivatives that retain anti-rhabdomyosarcoma activity without anti-hypertensive effects.

All-optical linearized Mach-Zehnder modulator.

A practical, broadband, all-optical linearization concept for a Mach-Zehnder modulator (MZM) is proposed and demonstrated. The unique transmitter design includes an amplitude modulated (AM) standard MZM with two optical outputs, where the alternative (or complimentary) output is combined with the laser carrier to create a linearizing optical local oscillator, which when coherently combined with the AM signal fully cancels 3rd order intermodulation distortion components. Using this scheme, record linearity is achieved for a non-amplified RF photonic link, with spurious free dynamic range (SFDR) of 118.5 dB.Hz2/3 and 123 dB.Hz2/3 for single and dual fiber/photodetector schemes.

Gatekeeper training for friends and family of individuals at risk of suicide: A systematic review.

AIMS: Gatekeeper training (GKT) is an important suicide prevention strategy. Studies have evaluated the effectiveness of GKT in different populations, often neglecting family and friends who play a vital role in caring for people with suicide risk. This review evaluated GKT programs targeting family and friends to determine their effectiveness in this specific population. METHODS: Academic databases were searched for studies on GKT programs. Programs involving family and friends caring for people with suicide risk were assessed for any impact on knowledge, self-efficacy, attitudes, and suicide prevention skills. RESULTS: Seventeen studies were reviewed. GKT showed significant gains on outcomes of interest. Three studies targeted family and friends, with one involving them in program creation and conduction and another adjusting the program after their input. CONCLUSIONS: GKT programs have potentially positive effects on family and friends caring for people with suicide risk. Few programs address the specific needs of this group, and programs adapted specifically for them are scarce. Future program development recommendations are discussed.

K2p 3.1 protein is expressed as a transmural gradient across the rat left ventricular free wall.

INTRODUCTION: K2p 3.1, also known as TASK-1, is a twin-pore acid-sensitive repolarizing K+ channel, responsible for a background potassium current that significantly contributes to setting the resting membrane potential of cardiac myocytes. Inhibition of IK2p3.1 alters cardiac repolarization and is proarrhythmogenic. In this study, we have examined the expression of K2p 3.1 and function of this channel in tissue and myocytes from across the left ventricular free wall. METHODS AND RESULTS: Using fluorescence immunocytochemistry, the expression of K2p 3.1 protein in myocytes from the subendocardial region was found to be twice (205% ± 13.5%) that found in myocytes from the subepicardial region of the left ventricle (100% ± 5.3%). The left ventricular free wall exhibited a marked transmural gradient of K2p 3.1 protein expression. Western blot analysis confirmed significantly higher K2p 3.1 protein expression in subendocardial tissue (156% ± 2.5%) than subepicardial tissue (100% ± 5.0%). However, there was no difference in K2p 3.1 messenger RNA expression. Whole-cell patch clamp identified IK2p3.1 current density to be significantly greater in myocytes isolated from the subendocardium (7.66 ± 0.53 pA/pF) compared with those from the subepicardium (3.47 ± 0.74 pA/pF). CONCLUSIONS: This is the first study to identify a transmural gradient of K2p 3.1 in the left ventricle. This gradient has implications for understanding ventricular arrhythmogenesis under conditions of ischemia but also in response to other modulatory factors, such as adrenergic stimulation and the presence of anesthetics that inhibits or activates this channel.

Examining spatiotemporal variability of urban particulate matter and application of high-time resolution data from a network of low-cost air pollution sensors.

Traditional air monitoring approaches using regulatory monitors have historically been used to assess regional-scale trends in air pollutants across large geographical areas. Recent advances in air pollution sensor technologies could provide additional information about nearby sources, support the siting of regulatory monitoring stations, and improve our knowledge of finer-scale spatiotemporal variation of ambient air pollutants and their associated health effects. Sensors are now being developed that are much smaller and lower cost than traditional ambient air monitoring systems and are capable of being deployed as a network to provide greater coverage of a given area. The CitySpace project conducted by the US EPA and the Shelby County Health Department included the deployment of a network of 17 sensor pods using Alphasense OPC-N2 particulate matter (PM) sensors integrated with meteorological sensors in Memphis, TN for six months. Sensor pods were collocated with a federal equivalent method (FEM) tapered element oscillating microbalance (TEOM) monitor both before and after the primary study period. Six of the sensor pods were found to meet the data quality objective (DQO) of coefficient of determination (R2) greater than 0.5 when collocated with the TEOM. Seven pods were decommissioned before the end of the study due to mechanical failure. The six pods meeting the DQO were used to examine the spatiotemporal variability of fine PM (PM2.5) across the Memphis area. One site was found to have higher relative PM2.5 concentrations when compared to the other sites in the network. The 1-min data from this sensor pod were evaluated to quantify the regional urban background and local-scale contributions to PM2.5 at that monitoring location. This method found that approximately 20% of the PM2.5 was attributed to local sources at this location, compared to 9% at a local regulatory monitoring site. Additionally, the 1-min data were combined with 1-min wind speed and wind direction data to examine potential sources in the area using the nonparametric trajectory analysis (NTA) technique. This method geographically identified local source areas that contributed to the measured concentrations at the high reading sensor location throughout the course of the study.

Neuropsychiatric manifestations of Sydenham's chorea: a systematic review.

AIM: Sydenham's chorea is a post-streptococcal, autoimmune, neuropsychiatric movement disorder. Sydenham's chorea is a major criterion for diagnosis of acute rheumatic fever with the implication of potential long-term sequelae including cardiac complications. It is well established that there is psychiatric comorbidity in Sydenham's chorea, but there are variations in the literature regarding the nature and prevalence of psychiatric diagnoses associated with Sydenham's chorea. The aim of this review was to systematically evaluate the evidence for psychiatric symptoms presenting with Sydenham's chorea. Knowledge of comorbid psychiatric symptomatology will support early diagnosis and treatment, leading to improved long-term outcomes for children with Sydenham's chorea. METHOD: The study used a systematic search strategy, using MEDLINE, MEDLINE in Process, EMBASE, and The Cochrane Library. Abstracts were screened to identify relevant papers which were then assessed further. Eligible papers were summarized. RESULTS: A total of 1429 abstracts of relevant studies were found, and 49 papers reporting neuropsychiatric symptoms in Sydenham's chorea were summarized. Obsessive-compulsive disorder was the most commonly studied, and hence reported, neuropsychiatric symptom in children with Sydenham's chorea. The studies analysed used a variety of tools to identify affected children and used different methods for analysing results. Attention-deficit-hyperactivity disorder, affective disorders, tic disorders, executive function disturbances, and psychotic features were also reported as comorbidities. INTERPRETATION: There is good evidence of neuropsychiatric comorbidities in Sydenham's chorea. In countries with a high prevalence of rheumatic fever, the early recognition of salient cognitive and psychiatric symptoms may aid in the management of Sydenham's chorea.

Insights from analysis for harmful and potentially harmful constituents (HPHCs) in tobacco products.

A total of 20 commercial cigarette and 16 commercial smokeless tobacco products were assayed for 96 compounds listed as harmful and potentially harmful constituents (HPHCs) by the US Food and Drug Administration. For each product, a single lot was used for all testing. Both International Organization for Standardization and Health Canada smoking regimens were used for cigarette testing. For those HPHCs detected, measured levels were consistent with levels reported in the literature, however substantial assay variability (measured as average relative standard deviation) was found for most results. Using an abbreviated list of HPHCs, statistically significant differences for most of these HPHCs occurred when results were obtained 4-6months apart (i.e., temporal variability). The assay variability and temporal variability demonstrate the need for standardized analytical methods with defined repeatability and reproducibility for each HPHC using certified reference standards. Temporal variability also means that simple conventional comparisons, such as two-sample t-tests, are inappropriate for comparing products tested at different points in time from the same laboratory or from different laboratories. Until capable laboratories use standardized assays with established repeatability, reproducibility, and certified reference standards, the resulting HPHC data will be unreliable for product comparisons or other decision making in regulatory science.

Treatments and Outcomes Among Patients with Sydenham Chorea: A Meta-Analysis.

Importance: Sydenham chorea is the most common acquired chorea of childhood worldwide; however, treatment is limited by a lack of high-quality evidence. Objectives: To evaluate historical changes in the clinical characteristics of Sydenham chorea and identify clinical and treatment factors at disease onset associated with chorea duration, relapsing disease course, and functional outcome. Data Sources: The systematic search for this meta-analysis was conducted in PubMed, Embase, CINAHL, Cochrane Library, and LILACS databases and registers of clinical trials from inception to November 1, 2022 (search terms: [Sydenham OR Sydenham's OR rheumatic OR minor] AND chorea). Study Selection: Published articles that included patients with a final diagnosis of Sydenham chorea (in selected languages). Data Extraction and Synthesis: This study followed the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) reporting guideline. Individual patient data on clinical characteristics, treatments, chorea duration, relapse, and final outcome were extracted. Data from patients in the modern era (1945 through 2022) were entered into multivariable models and stratified by corticosteroid duration for survival analysis of chorea duration. Main Outcomes and Measures: The planned study outcomes were chorea duration at onset, monophasic course (absence of relapse after ≥24 months), and functional outcome (poor: modified Rankin Scale score 2-6 or persisting chorea, psychiatric, or behavioral symptoms at final follow-up after ≥6 months; good: modified Rankin Scale score 0-1 and no chorea, psychiatric, or behavioral symptoms at final follow-up). Results: In total, 1479 patients were included (from 307 articles), 1325 since 1945 (median [IQR] age at onset, 10 [8-13] years; 875 of 1272 female [68.8%]). Immunotherapy was associated with shorter chorea duration (hazard ratio for chorea resolution, 1.51 [95% CI, 1.05-2.19]; P = .03). The median chorea duration in patients receiving 1 or more months of corticosteroids was 1.2 months (95% CI, 1.2-2.0) vs 2.8 months (95% CI, 2.0-3.0) for patients receiving none (P = .004). Treatment factors associated with monophasic disease course were antibiotics (odds ratio [OR] for relapse, 0.28 [95% CI, 0.09-0.85]; P = .02), corticosteroids (OR, 0.32 [95% CI, 0.15-0.67]; P = .003), and sodium valproate (OR, 0.33 [95% CI, 0.15-0.71]; P = .004). Patients receiving at least 1 month of corticosteroids had significantly lower odds of relapsing course (OR, 0.10 [95% CI, 0.04-0.25]; P < .001). No treatment factor was associated with good functional outcome. Conclusions and Relevance: In this meta-analysis of treatments and outcomes in patients with Sydenham chorea, immunotherapy, in particular corticosteroid treatment, was associated with faster resolution of chorea. Antibiotics, corticosteroids and sodium valproate were associated with a monophasic disease course. This synthesis of retrospective data should support the development of evidence-based treatment guidelines for patients with Sydenham chorea.

Azetidines Kill Multidrug-Resistant Mycobacterium tuberculosis without Detectable Resistance by Blocking Mycolate Assembly.

Tuberculosis (TB) is the leading cause of global morbidity and mortality resulting from infectious disease, with over 10.6 million new cases and 1.4 million deaths in 2021. This global emergency is exacerbated by the emergence of multidrug-resistant MDR-TB and extensively drug-resistant XDR-TB; therefore, new drugs and new drug targets are urgently required. From a whole cell phenotypic screen, a series of azetidines derivatives termed BGAz, which elicit potent bactericidal activity with MIC99 values <10 µM against drug-sensitive Mycobacterium tuberculosis and MDR-TB, were identified. These compounds demonstrate no detectable drug resistance. The mode of action and target deconvolution studies suggest that these compounds inhibit mycobacterial growth by interfering with cell envelope biogenesis, specifically late-stage mycolic acid biosynthesis. Transcriptomic analysis demonstrates that the BGAz compounds tested display a mode of action distinct from the existing mycobacterial cell wall inhibitors. In addition, the compounds tested exhibit toxicological and PK/PD profiles that pave the way for their development as antitubercular chemotherapies.

Preservation of cardiomyocytes from the adult heart.

Cardiomyocytes represent one of the most useful models to conduct cardiac research. A single adult heart yields millions of cardiomyocytes, but these cells do not survive for long after isolation. We aimed to determine whether inhibition of myosin II ATPase that is essential for muscle contraction may preserve fully differentiated adult cardiomyocytes. Using inhibitors of the myosin II ATPase, blebbistatin and N-benzyl-p-toluene sulphonamide (BTS), we preserved freshly isolated fully differentiated adult primary cardiomyocytes that were stored at a refrigerated temperature. Specifically, preserved cardiomyocytes stayed viable for a 2-week period with a stable expression of cardiac genes and retained the expression of key markers characteristic of cardiomyocytes. Furthermore, voltage-clamp, action potential, calcium transient and contractility studies confirmed that the preserved cardiomyocytes are comparable to freshly isolated cells. Long-term exposure of preserved cardiomyocytes to four tyrosine kinase inhibitors, sunitinib malate, dasatinib, sorafenib tosylate and imatinib mesylate, revealed their potential to induce cardiac toxicity that was manifested with a decrease in contractility and induction of cell death, but this toxicity was not observed in acute experiments conducted over the time course amenable to freshly prepared cardiomyocytes. This study introduces the concept that the inhibition of myosin II ATPase safeguards the structure and function of fully differentiated adult cardiomyocytes. The fact that these preserved cardiomyocytes can be used for numerous days after preparation makes them a robust and versatile tool in cardiac research and allows the investigation of long-term exposure to novel drugs on cardiomyocyte function.

Adults with attention-deficit hyperactivity disorder--diagnosis or normality?

Attention-deficit hyperactivity disorder in adults evokes extreme responses within British psychiatrists, because its diagnostic validity and pharmacological treatments are heavily contested. We propose a model that accommodates apparently divergent evidence, and provides a clinical framework for clinicians and patients, allowing safe, responsible and ethically balanced clinical practice.

An integrated approach for early in vitro seizure prediction utilizing hiPSC neurons and human ion channel assays.

Seizure liability remains a significant cause of attrition throughout drug development. Advances in stem cell biology coupled with an increased understanding of the role of ion channels in seizure offer an opportunity for a new paradigm in screening. We assessed the activity of 15 pro-seizurogenic compounds (7 CNS active therapies, 4 GABA receptor antagonists, and 4 other reported seizurogenic compounds) using automated electrophysiology against a panel of 14 ion channels (Nav1.1, Nav1.2, Nav1.6, Kv7.2/7.3, Kv7.3/7.5, Kv1.1, Kv4.2, KCa4.1, Kv2.1, Kv3.1, KCa1.1, GABA α1ß2γ2, nicotinic α4ß2, NMDA 1/2A). These were selected based on linkage to seizure in genetic/pharmacological studies. Fourteen compounds demonstrated at least one "hit" against the seizure panel and 11 compounds inhibited 2 or more ion channels. Next, we assessed the impact of the 15 compounds on electrical signaling using human-induced pluripotent stem cell neurons in microelectrode array (MEA). The CNS active therapies (amoxapine, bupropion, chlorpromazine, clozapine, diphenhydramine, paroxetine, quetiapine) all caused characteristic changes to electrical activity in key parameters indicative of seizure such as network burst frequency and duration. The GABA antagonist picrotoxin increased all parameters, but the antibiotics amoxicillin and enoxacin only showed minimal changes. Acetaminophen, included as a negative control, caused no changes in any of the parameters assessed. Overall, pro-seizurogenic compounds showed a distinct fingerprint in the ion channel/MEA panel. These studies highlight the potential utility of an integrated in vitro approach for early seizure prediction to provide mechanistic information and to support optimal drug design in early development, saving time and resources.

Childhood/adolescent Sydenham's chorea in the UK and Ireland: a BPSU/CAPSS surveillance study.

OBJECTIVE: To conduct the first prospective surveillance study of Sydenham's chorea (SC) in the UK and Ireland, and to describe the current paediatric and child psychiatric service-related incidence, presentation and management of SC in children and young people aged 0-16 years. DESIGN: Surveillance study of first presentations of SC reported by paediatricians via the British Paediatric Surveillance Unit (BPSU) and all presentations of SC reported by child and adolescent psychiatrists through the Child and Adolescent Psychiatry Surveillance System (CAPSS). RESULTS: Over 24 months from November 2018, 72 reports were made via BPSU, of which 43 met the surveillance case definition of being eligible cases of suspected or confirmed SC. This translates to an estimated paediatric service-related incidence rate of new SC cases of 0.16 per 100 000 children aged 0-16 per year in the UK. No reports were made via CAPSS over the 18-month reporting period, although over 75% of BPSU cases presented with emotional and/or behavioural symptoms. Almost all cases were prescribed courses of antibiotics of varying duration, and around a quarter of cases (22%) received immunomodulatory treatment. CONCLUSIONS: SC remains a rare condition in the UK and Ireland but has not disappeared. Our findings emphasise the impact that the condition can have on children's functioning and confirm that paediatricians and child psychiatrists should remain vigilant to its presenting features, which commonly include emotional and behavioural symptoms. There is a further need for development of consensus around identification, diagnosis and management across child health settings.

Discriminatory power of standard toxicity assays used to evaluate ingredients added to cigarettes.

A tiered approach for testing ingredients in a cigarette matrix was developed and includes chemical-analytical testing and a standard battery of biological toxicity assays. These assays were adapted for comparative evaluation of mainstream smoke from experimental cigarettes with or without ingredients at various inclusion levels. This adaptation to test cigarette mainstream smoke may impact assay response. Since it is difficult to a priori determine discriminatory power, it was evaluated using a large experimental dataset from a multi-year program of cigarette ingredient testing performed at two separate laboratories. A statistical method, minimum detectable difference (MDD), was used as a measure of assay discriminatory power. MDD of cigarette smoke constituents ranged from 6% to 29% of the average. Salmonella mutagenicity and cytotoxicity test MDDs ranged from 20% to 81% and 18% to 49%, respectively. Body weight gain in 90-day nose-only inhalation studies yielded an MDD of 30-40%. Histopathological findings with severity scores between 0.5 and 1.5 had the lowest MDDs of 23% and higher. In general, discriminatory power decreased with increasing biological complexity and toxicological relevance of the assay. Beyond statistical analysis, however, a weight-of-the-evidence analysis by experienced researchers is required for toxicological assessment of a cigarette ingredient.

Classification, prevalence and integrated care for neurodevelopmental and child mental health disorders: A brief overview for paediatricians.

'Neurodevelopmental disorders' comprise a group of congenital or acquired long-term conditions that are attributed to disturbance of the brain and or neuromuscular system and create functional limitations, including autism spectrum disorder, attention deficit/ hyperactivity disorder, tic disorder/ Tourette's syndrome, developmental language disorders and intellectual disability. Cerebral palsy and epilepsy are often associated with these conditions within the broader framework of paediatric neurodisability. Co-occurrence with each other and with other mental health disorders including anxiety and mood disorders and behavioural disturbance is often the norm. Together these are referred to as neurodevelopmental, emotional, behavioural, and intellectual disorders (NDEBIDs) in this paper. Varying prevalence rates for NDEBID have been reported in developed countries, up to 15%, based on varying methodologies and definitions. NDEBIDs are commonly managed by either child health paediatricians or child/ adolescent mental health (CAMH) professionals, working within multidisciplinary teams alongside social care, education, allied healthcare practitioners and voluntary sector. Fragmented services are common problems for children and young people with multi-morbidity, and often complicated by sub-threshold diagnoses. Despite repeated reviews, limited consensus among clinicians about classification of the various NDEBIDs may hamper service improvement based upon research. The recently developed "Mental, Behavioural and Neurodevelopmental disorder" chapter of the International Classification of Diseases-11 offers a way forward. In this narrative review we search the extant literature and discussed a brief overview of the aetiology and prevalence of NDEBID, enumerate common problems associated with current classification systems and provide recommendations for a more integrated approach to the nosology and clinical care of these related conditions.

Association with {beta}-COP regulates the trafficking of the newly synthesized Na,K-ATPase.

Plasma membrane expression of the Na,K-ATPase requires assembly of its α- and ß-subunits. Using a novel labeling technique to identify Na,K-ATPase partner proteins, we detected an interaction between the Na,K-ATPase α-subunit and the coat protein, ß-COP, a component of the COP-I complex. When expressed in the absence of the Na,K-ATPase ß-subunit, the Na,K-ATPase α-subunit interacts with ß-COP, is retained in the endoplasmic reticulum, and is targeted for degradation. In the presence of the Na,K-ATPase ß-subunit, the α-subunit does not interact with ß-COP and traffics to the plasma membrane. Pulse-chase experiments demonstrate that in cells expressing both the Na,K-ATPase α- and ß-subunits, newly synthesized α-subunit associates with ß-COP immediately after its synthesis but that this interaction does not constitute an obligate intermediate in the assembly of the α- and ß-subunits to form the pump holoenzyme. The interaction with ß-COP was reduced by mutating a dibasic motif at Lys(54) in the Na,K-ATPase α-subunit. This mutant α-subunit is not retained in the endoplasmic reticulum and reaches the plasma membrane, even in the absence of Na,K-ATPase ß-subunit expression. Although the Lys(54) α-subunit reaches the cell surface without need for ß-subunit assembly, it is only functional as an ion-transporting ATPase in the presence of the ß-subunit.

Hepatic lipid peroxidation and cytochrome P-450 2E1 in pediatric nonalcoholic fatty liver disease and its subtypes.

GOAL: To compare hepatic lipid peroxidation and cytochrome P-450 2E1 (CYP2E1) protein content in liver biopsies from children with nonalcoholic fatty liver disease (NAFLD) and 2 control groups. BACKGROUND: Elevated hepatic lipid peroxidation resulting from increased hepatic CYP2E1 enzyme activity is involved in the pathogenesis of NAFLD and nonalcoholic steatohepatitis (NASH) in adults, but studies in children are lacking. STUDY: Liver biopsies from 59 children with NAFLD (49 with NASH), 10 children with normal liver histology, and 9 children with mild chronic hepatitis C (HCV) infection were examined. Hepatic malondialdehyde (a measure of lipid peroxidation) levels and CYP2E1 protein content were quantitated, as a percentage of the total area, by immunohistochemical staining of liver biopsy material followed by digital image quantitation. RESULTS: Lipid peroxidation was significantly greater in NAFLD liver biopsies (46.7 ± 20.8%) compared with biopsies from children with normal liver histology (7.6 ± 9.4%; P<0.001) or HCV infection (7.7 ± 7.6%; P<0.001). However, hepatic CYP2E1 expression was not different across the NAFLD, normal liver histology, and HCV groups (60.7 ± 8.7%, 53.5 ± 10.7%, and 60.0 ± 11.9%, respectively; P=0.116). Among children with NAFLD, lipid peroxidation and CYP2E1 protein content did not differ between biopsies with and without NASH. Body mass index was independently associated with hepatic lipid peroxidation levels (r=0.549; P<0.001). CONCLUSIONS: Hepatic lipid peroxidation is increased in children with NAFLD but this is not related to hepatic CYP2E1 expression. No difference in lipid peroxidation in pediatric NAFLD versus NASH argues against a role in disease progression.

Insights from a multi-year program designed to test the impact of ingredients on mainstream cigarette smoke toxicity.

CONTEXT: Cigarette tobacco ingredients may alter the distribution of chemical constituents present in smoke. When considering the toxicological relevance of potential ingredient-related effects on chemical and biological measurements assessing cigarette smoke toxicity, it is critical to understand the intrinsic variability of tobacco and cigarette smoke that is influenced by the environmental conditions during growing, agricultural practices during preparation, cigarette manufacturing tolerances, and stability of the assay methods. OBJECTIVE: To understand possible effects of ingredients on cigarette smoke toxicity, various chemical and biological endpoints were measured in smoke from experimental cigarettes (added ingredient) to the intrinsic variability of control cigarettes (no added ingredient). MATERIALS AND METHODS: Data were collected during a multi-year program testing a variety of cigarette ingredients from several chemical classes. Chemical analysis of mainstream cigarette smoke,and biological procedures (Salmonella mutagenicity, cytotoxicity, and smoke inhalation) were performed using validated and controlled laboratory methods. The within-study and temporal variation of control cigarettes manufactured in parallel with experimental cigarettes was calculated and used to measure intrinsic variability. RESULTS: The overwhelming majority of data generated from experimental cigarettes fell within the experiment variability represented by the pooled standard error of the entire multi-year dataset for the control cigarettes. CONCLUSION: The results of this evaluation add to a growing body of the literature regarding a weight of evidence assessment of cigarette ingredient toxicity. When assessed against the variability of assay methodology, natural agricultural change, and manufacturing control, the ingredients studied here demonstrated little relevant influence on the mainstream cigarette smoke toxicity endpoints measured.