Effect of Rythmol (propafenone HCl) administration during pregnancy in Wistar rats.

Propafenone is a well-known Class 1C antiarrhythmic agent that has sodium channel blocking properties as well as the ability to block 13 other channels and a modest calcium antagonistic effect. Propafenone has a profound electrophysiologic effect on auxiliary atrioventricular circuits and in patients with atrioventricular nodal reentry tachycardia can obstruct conduction in the fast conducting pathway. Furthermore, propafenone is less likely than other Class 1C drugs to cause proarrhythmia. However, although this medicine can pass through the placenta, the effects during pregnancy remain unknown. Here, we investigated the potential teratogenic and genotoxic effects of Rythmol during rat development. Pregnant Wistar rats received 46.25 mg/kg body weight of propafenone daily by gavage from Gestation Day (GD) 5 to GD 19. At GD 20, the dams were dissected, and their fetuses were assessed via morphologic, skeletal, and histologic investigation. In addition, a comet assay was used to measure DNA impairment of fetal skull osteocytes and hepatic cells. The study showed that propafenone treatment of pregnant rats led to a marked decrease in gravid uterine weight, number of implants/litter, number of viable fetuses, and bodyweight of fetuses but a clear increase in placental weight and placental index in the treated group. Frequent morphologic abnormalities and severe ossification deficiency in the cranium bones were observed in the treatment group. Various histopathological changes were observed in the liver, kidney, and brain tissues of maternally treated fetuses. Similarly, propafenone induced DNA damage to examined samples. Thus, our study indicates that propafenone may be embryotoxic in humans.

Anti-PD-1/PD-L1 therapy augments lenvatinib's efficacy by favorably altering the immune microenvironment of murine anaplastic thyroid cancer.

Patients with anaplastic thyroid cancer (ATC) have an extremely poor prognosis despite multimodal therapy with surgery and chemoradiation. Lenvatinib, a multi-targeted tyrosine kinase inhibitor, as well as checkpoint inhibitors targeting the programmed cell death pathway, have proven effective in some patients with advanced thyroid cancer. Combination of these therapies is a potential means to boost effectiveness and minimize treatment resistance in ATC. We utilized our novel immunocompetent murine model of orthotopic ATC to demonstrate that lenvatinib led to significant tumor shrinkage and increased survival, while combination therapy led to dramatic improvements in both. Lenvatinib monotherapy increased tumor-infiltrating macrophages, CD8+ T-cells, regulatory T-cells, and most notably, polymorphonuclear myeloid derived suppressor cells (PMN-MDSCs). While both combination therapies led to further increases in CD8+ T-cells, only the lenvatinib and anti-PD-1 combination decreased PMN-MDSCs. PMN-MDSC expansion was also seen in the blood of mice and one patient receiving lenvatinib therapy for ATC. RNA-Seq of the ATC cell line used in our mouse model demonstrated that lenvatinib has multifaceted effects on angiogenesis, response to hypoxia, the epithelial-to-mesenchymal transition, and on multiple pathways implicated in inflammation and host immunity. Combination of lenvatinib with anti-Gr-1 antibody ameliorated lenvatinib's expansion of MDSCs and significantly improved lenvatinib's anti-tumor effect. These data suggest that MDSCs play a negative role in ATC's response to lenvatinib and support future study of their role as a potential biomarker and treatment target.

Combinations of BRAF inhibitor and anti-PD-1/PD-L1 antibody improve survival and tumour immunity in an immunocompetent model of orthotopic murine anaplastic thyroid cancer.

BACKGROUND: Patients with anaplastic thyroid cancer (ATC) have an extremely poor prognosis despite aggressive multimodal therapy. ATC has a high prevalence of BRAFV600E mutations and is associated with an immunosuppressive microenvironment; we previously demonstrated that the combination of BRAF inhibitor and checkpoint inhibitor immunotherapy synergistically reduce tumour volume in an immunocompetent mouse model of orthotopic ATC. METHODS: We again utilised our mouse model of ATC to assess the combination of BRAFV600E inhibitor PLX4720 and anti-PD-L1 or anti-PD-1 antibody on survival, and performed immune cell profiling of lymphoid and myeloid-lineage cells during maximal treatment response and tumour regrowth. RESULTS: Combination therapy dramatically improved mouse survival. Maximal tumour reduction was associated with increases in the number and cytotoxicity of CD8+ T cells and NK cells, as well as increases in mostly M1-polarised tumour-associated macrophages (TAM) and decreases in myeloid-derived suppressor-like cells. Regrowth of tumour occurred after 2-3 weeks of ongoing combination therapy, and was most significantly associated with decreased TAMs and a dramatic increase in M2-polarisation. CONCLUSIONS: Combination of PLX4720 and anti-PD-L1/PD-1 antibody dramatically reduced tumour volume, prolonged survival and improved the anti-tumour immune profile in murine ATC. Tumour growth inevitably recurred and demonstrated re-emergence of an immunosuppressive tumour microenvironment.

Awareness and determinants of electronic cigarette use among Finnish adolescents in 2013: a population-based study.

BACKGROUND: A wide range of electronic cigarettes (e-cigarettes) are now on the market. We studied e-cigarette awareness and use, determinants and sources of e-cigarettes, the e-liquids used in them and exposure to e-cigarette advertisements among adolescents in Finland. Among smokers, we studied the association of e-cigarette use and interest in smoking cessation. METHOD: Data were obtained from a national survey of 12-18-year-old Finnish adolescents in 2013 (N=3535, response rate 38%). Descriptive statistics and logistic regression analysis were used. RESULTS: Of the respondents, 85.3% knew what e-cigarettes were; 17.4% had tried them. E-liquids with nicotine were used most often (65.7%); also those who had never tried conventional cigarettes had used them. Of e-cigarette ever users, 8.3% had never tried smoking. Parents' high level of education, being in employment, and intact family protected against children's e-cigarette use. In the final model, daily smoking (OR 41.35; 95% CI 25.2 to 67.8), snus use (2.96; 2.4-4.0), waterpipe use (2.21; 1.6-3.0), children's vocational education (2.06; 1.4-3.1) and poor school performance (1.92; 1.4-3.0) were associated with e-cigarette experimentation. Those smokers with most experience of e-cigarettes were least likely to consider smoking cessation. CONCLUSIONS: Awareness and experimentation with e-cigarettes are high among adolescents, especially in older age groups and boys. Nicotine e-liquids are easy to acquire for youth. Having similar risk factors, e-cigarette use seems to follow the model of conventional smoking initiation. Among adolescent smokers, use of e-cigarettes does not clearly relate to interest in smoking cessation. Preventive policies are needed to protect the youth.

Multiple parallel cell lineages in the developing mammalian cerebral cortex.

Cortical neurogenesis follows a simple lineage: apical radial glia cells (RGCs) generate basal progenitors, and these produce neurons. How this occurs in species with expanded germinal zones and a folded cortex, such as human, remains unclear. We used single-cell RNA sequencing from individual cortical germinal zones in ferret and barcoded lineage tracking to determine the molecular diversity of progenitor cells and their lineages. We identified multiple RGC classes that initiate parallel lineages, converging onto a common class of newborn neuron. Parallel RGC classes and transcriptomic trajectories were repeated across germinal zones and conserved in ferret and human, but not in mouse. Neurons followed parallel differentiation trajectories in the gyrus and sulcus, with different expressions of human cortical malformation genes. Progenitor cell lineage multiplicity is conserved in the folded mammalian cerebral cortex.

Immigration-related Legal Collaboration in Primary Care: Evaluating Impact on Knowledge and Well-being.

OBJECTIVES: To evaluate the impact of embedding an immigration attorney in a primary care clinic to address immigration-related legal needs. METHODS: We conducted a mixed-methods study of 42 legal clinic participants from May 2019-February 2020. Measures included psychological distress, understanding of legal options, and self-rated general health collected prior to, following, and 60-90 days after consultation. RESULTS: There was significant improvement in participants' understanding of their legal immigration options pre- (4.9, SD 2.9) and post-consult (8.6, SD 2.1), and 60 days later (7.0, SD 2.8) (F=11.0, p<.05), but self-rated health scores and distress did not significantly improve, although there was a high loss-to-follow up rate at 60 days (42.8%). Qualitative results underscored the interconnectedness of immigration status and health. DISCUSSION: Embedding immigration legal services in primary care improved patients' understanding of immigration-related legal options, although successfully mitigating the health impacts of vulnerable immigration status may take broader societal interventions.

Adolescents' Perceptions of Harmfulness of Tobacco and Tobacco-like Products in Finland.

During the recent years, new tobacco and tobacco-like products, e.g., e-cigarettes, have emerged on the market. Adolescents often underestimate health risks in general, including those concerning tobacco. Little is known of adolescents' perceptions of health risks of the newer products. Our paper compares adolescents' perceptions of harmfulness of cigarettes, e-cigarettes, snus, water pipes, and nicotine in Finland, a country with a long history of strict tobacco control policy. Online surveys to nationally representative samples of 12-18-year-olds were conducted in 2017 and 2019, with 7578 answering the surveys. Only 3% of boys and 2% of girls did not agree that cigarettes are harmful to health. The percentages were slightly higher for snus (6% and 3%, respectively) and nicotine (12%, 8%) but much higher for e-cigarettes (30%, 22%) and water pipes (36%, 38%). Those who used the product, whose parents were smokers or had lower education, and whose school performance was lower, less often agreed with the harmful health effects of the products. Our results showed that adolescents understood the harmfulness of older tobacco products better than the harmfulness of the newer ones. Our results also showed the need to strengthen health education and fix adolescents' misperceptions of the health effects of the newer products.

The role of parents, friends and teachers in adolescents' cigarette smoking and tombak dipping in Sudan.

OBJECTIVE: To assess the influence of smoking and tombak (local smokeless tobacco) dipping by parents, teachers and friends on cigarette smoking and tombak dipping by school-going Sudanese adolescents. METHODS: This was a school-based cross-sectional survey was conducted in 2005-2006. Logistic regression was used for the analysis. A total of 4277 Sudanese school-going adolescents (aged 11-17 years) from 23 schools who completed an anonymous self-administered questionnaire on the use of tobacco products. Main outcome measures were self-reported tobacco use during the previous month defined current tobacco use. Ever smoking, tombak dipping and other tobacco products were also considered as outcomes. RESULTS: After adjusting for sex, age and school grade, adolescents' smoking habits were strongly associated with the habit in their parents and friends and, more weakly, with tombak dipping by teachers. When adjusted for each other, the association with smoking in friends was unaffected and remained significant (prevalence OR (POR) of having ever smoked was 1.94, 95% CI 1.64 to 2.29; OR of being current smoker was 3.77, 95% CI 2.80 to 5.07). Tobacco smoking in friends was positively associated with adolescents ever tombak dipping (POR 1.81, 95% CI 1.41 to 2.33) and current dipping (OR 3.33, 95% CI 2.20 to 5.05). The association with parental habits was reduced but still significantly elevated. Tombak dipping by teachers was only associated with adolescents ever tobacco smoking. CONCLUSIONS: Tobacco use by parents, teachers and friends was associated with adolescents' tobacco habits. The influence of friends was the strongest. In developing programmes against adolescents' tobacco habits, there is need to target the influence of these 'significant others'. Sudan needs to develop and implement comprehensive anti-smoking and anti-tombak dipping legislation to reduce the growing prevalence of such habits.

The Extracellular Matrix in the Evolution of Cortical Development and Folding.

The evolution of the mammalian cerebral cortex leading to humans involved a remarkable sophistication of developmental mechanisms. Specific adaptations of progenitor cell proliferation and neuronal migration mechanisms have been proposed to play major roles in this evolution of neocortical development. One of the central elements influencing neocortex development is the extracellular matrix (ECM). The ECM provides both a structural framework during tissue formation and to present signaling molecules to cells, which directly influences cell behavior and movement. Here we review recent advances in the understanding of the role of ECM molecules on progenitor cell proliferation and neuronal migration, and how these contribute to cerebral cortex expansion and folding. We discuss how transcriptomic studies in human, ferret and mouse identify components of ECM as being candidate key players in cortex expansion during development and evolution. Then we focus on recent functional studies showing that ECM components regulate cortical progenitor cell proliferation, neuron migration and the mechanical properties of the developing cortex. Finally, we discuss how these features differ between lissencephalic and gyrencephalic species, and how the molecular evolution of ECM components and their expression profiles may have been fundamental in the emergence and evolution of cortex folding across mammalian phylogeny.

School Smoking Policies and Health Science Students' Use of Cigarettes, Shisha, and Dipping Tombak in Sudan.

The relationship between school smoking policies and students' tobacco use is ambiguous, and little is known about the effect of these policies in low- and middle-income countries. This study was designed to assess the effects of schools' smoking policies and the exposure to residential smoking on cigarette smoking and the use of different kinds of tobacco products by Health Science students. Self-reports of cigarette smoking, use of shisha (smoking of fruits-mixed tobacco using a bowl and a connected hose); dipping tombak (local smokeless tobacco that users usually place inside oral cavity in the groove behind the lower lip), and tobacco use on school premises are analyzed. A cross-sectional survey was carried out using a modified self-report questionnaire, originally developed by WHO, among a representative sample of 1,590 third-year HSS from 25 schools drawn from 13 universities, using a multi-stages sampling technique. The response rate was 100% for schools and 68% for students. A multilevel analysis was performed by nesting student-level in school-level variables. Results from the adjusted models revealed that, when students reported awareness of smoking restriction, they were more likely to be current smokers (OR = 2.91; 95% CI: 1.68-5.02; p = 0.021) and shisha users (OR = 2.17; 95% CI: 1.54-3.06; p = 0.021). Results from additional analysis performed among tobacco users only, showed increased risk of smokers and tombak dippers who smoked or dipped on school premises (OR = 2.38; 95% CI: 1.34-4.25; p = 0.003, OR = 2.60; 95% CI: 1.22-5.56; p = 0.013, respectively). Current smokers (OR = 3.12; 95% CI: 1.98-4.92; p = ≤ 0.001), ever smokers (OR = 1.66; 95% CI: 1.31-2.10; p = ≤ 0.001) and shisha users (OR = 1.73; 95% CI: 1.36-2.21; p = ≤ 0.001) were exposed to residential smoking on one or more days during the previous 7 days. High percentages of those who used any kind of tobacco products reported being aware of school smoking policies, indicating no clear evidence that school smoking policies had an effect on use of any of the mentioned tobacco products. The lack of compliance with school policies shows the need for further policy enforcement and sustainability, taking into account the effect of residential smoking and social influences.

Circulating BRAFV600E Levels Correlate with Treatment in Patients with Thyroid Carcinoma.

BACKGROUND: BRAFV600E is the most common mutation in papillary thyroid carcinoma (PTC) and can be associated with aggressive disease. Previously, a highly sensitive blood RNA-based BRAFV600E assay was reported. The objective of this study was to assess the correlation of BRAFV600E circulating tumor RNA levels with surgical and medical treatment. METHODS: Circulating BRAFV600E levels were assessed in (i) a murine model of undifferentiated (anaplastic) thyroid carcinoma with known BRAFV600E mutation undergoing BRAFV600E-inhibitor (BRAFi) treatment, and (ii) in 111 patients enrolled prior to thyroidectomy (n = 86) or treatment of advanced recurrent or metastatic PTC (n = 25). Blood samples were drawn for BRAFV600E analysis before and after treatment. Testing characteristics were assessed and positivity criteria optimized. Changes in blood BRAFV600E values were assessed and compared to clinical characteristics and response to therapy. RESULTS: In a murine model of anaplastic thyroid carcinoma with BRAFV600E mutation, blood BRAFV600E RNA correlated with tumor volume in animals treated with BRAFi. In tissue BRAFV600E-positive (n = 36) patients undergoing initial surgery for PTC, blood BRAFV600E levels declined postoperatively (median 370.0-178.5 fg/ng; p = 0.002). In four patients with metastatic or poorly differentiated thyroid carcinoma receiving targeted therapies, blood BRAFV600E declined following therapy and corresponded with radiographic evidence of partial response or stable disease. CONCLUSIONS: This study shows the correlation of blood BRAFV600E levels in response to treatment in both an established animal model of thyroid cancer and in patients with BRAFV600E-positive tumors with all stages of disease. This assay represents an alternative biomarker in patients with positive thyroglobulin antibodies, and tumors, which do not express thyroglobulin.

Widespread Chromosomal Losses and Mitochondrial DNA Alterations as Genetic Drivers in Hürthle Cell Carcinoma.

Hürthle cell carcinoma of the thyroid (HCC) is a form of thyroid cancer recalcitrant to radioiodine therapy that exhibits an accumulation of mitochondria. We performed whole-exome sequencing on a cohort of primary, recurrent, and metastatic tumors, and identified recurrent mutations in DAXX, TP53, NRAS, NF1, CDKN1A, ARHGAP35, and the TERT promoter. Parallel analysis of mtDNA revealed recurrent homoplasmic mutations in subunits of complex I of the electron transport chain. Analysis of DNA copy-number alterations uncovered widespread loss of chromosomes culminating in near-haploid chromosomal content in a large fraction of HCC, which was maintained during metastatic spread. This work uncovers a distinct molecular origin of HCC compared with other thyroid malignancies.

Inhibition of MAPKinase pathway sensitizes thyroid cancer cells to ABT-737 induced apoptosis.

Bcl2 family proteins play an important role in the resistance of thyroid cancer cells to apoptosis induced by chemotherapeutic drugs and targeted therapies. BH3-profiling of seven fresh primary papillary thyroid cancer (PTC) tumors showed dependence for survival on Bcl-xL (2/7), Bcl2 (2/7), and Mcl-1 (2/7), while the majority of thyroid cell lines were mainly dependent on Bcl-xL. Targeting Bcl2 family proteins with the BH3 mimetic, ABT-737, while simultaneously inhibiting ERK pathway proteins with PLX4720 and PD325901 was shown to induce significantly high apoptosis in the majority of cell lines (8505c, SW1736, HTh7, BCPAP) and moderate apoptosis in the TPC-1 cell line. In orthotopic thyroid cancer mouse models of 8505c and BCPAP, treatment with the triple drug combination reduced the size of the tumors and showed significantly higher numbers of cells undergoing apoptosis. This treatment increased the expression of pro-apoptotic protein Bim, while decreasing anti-apoptotic protein Mcl-1. Our results suggest that analyzing the results of BH3-profiling along with the mutational status of tumor can reveal an effective therapy for targeted, personalized treatment of aggressive thyroid cancer.

Combining BRAF inhibitor and anti PD-L1 antibody dramatically improves tumor regression and anti tumor immunity in an immunocompetent murine model of anaplastic thyroid cancer.

The interaction of programmed cell death-1 and its ligand is widely studied in cancer. Monoclonal antibodies blocking these molecules have had great success but little is known about them in thyroid cancer. We investigated the role of PD-L1 in thyroid cancer with respect to BRAF mutation and MAP kinase pathway activity and the effect of anti PD-L1 antibody therapy on tumor regression and intra-tumoral immune response alone or in combination with BRAF inhibitor (BRAFi). BRAFV600E cells showed significantly higher baseline expression of PD-L1 at mRNA and protein levels compared to BRAFWT cells. MEK inhibitor treatment resulted in a decrease of PD-L1 expression across all cell lines. BRAFi treatment decreased PD-L1 expression in BRAFV600E cells, but paradoxically increased its expression in BRAFWT cells. BRAFV600E mutated patients samples had a higher level of PD-L1 mRNA compared to BRAFWT (p=0.015). Immunocompetent mice (B6129SF1/J) implanted with syngeneic 3747 BRAFV600E/WT P53-/- murine tumor cells were randomized to control, PLX4720, anti PD-L1 antibody and their combination. In this model of aggressive thyroid cancer, control tumor volume reached 782.3±174.6mm3 at two weeks. The combination dramatically reduced tumor volume to 147.3±60.8, compared to PLX4720 (439.3±188.4 mm3, P=0.023) or PD-L1 antibody (716.7±62.1, P<0.001) alone. Immunohistochemistry analysis revealed intense CD8+ CTL infiltration and cytotoxicity and favorable CD8+:Treg ratio compared to each individual treatment. Our results show anti PD-L1 treatment potentiates the effect of BRAFi on tumor regression and intensifies anti tumor immune response in an immunocompetent model of ATC. Clinical trials of this therapeutic combination may be of benefit in patients with ATC.

Transmission of Smoking across Three Generations in Finland.

The influence of parents' smoking on children's smoking is well known, but few studies have examined the association between grandparents' and grandchildren's smoking. We studied the association between paternal and maternal grandparents' smoking and their grandchildren's tobacco use and assessed whether parents' smoking is a mediator in this process. Data were obtained from a national survey of 12-18-year-old Finns in 2013 (N = 3535, response rate 38%). Logistic regression and mediation analyses were used. Both boys and girls had higher odds for smoking experimentation, daily smoking and other tobacco or tobacco-like product use if their mother, father or any of the four grandparents were current or former smokers. When parents' and grandparents' smoking status were included in the same model, grandparents' smoking generally lost statistical significance. In the mediation analysis, 73% of the total effect of grandparents' smoking on grandchildren's daily smoking was mediated through parents' smoking, 64% on smoking experimentation and 63% on other tobacco or tobacco-like product use. The indirect effect of a mother's smoking was higher than that of a father's. To conclude, paternal and maternal grandparents' smoking increases grandchildren's tobacco use. The influence is mainly, but not completely, mediated through parents' smoking.

Molecularly defined unfolded protein response subclasses have distinct correlations with fatty liver disease in zebrafish.

The unfolded protein response (UPR) is a complex network of sensors and target genes that ensure efficient folding of secretory proteins in the endoplasmic reticulum (ER). UPR activation is mediated by three main sensors, which regulate the expression of hundreds of targets. UPR activation can result in outcomes ranging from enhanced cellular function to cell dysfunction and cell death. How this pathway causes such different outcomes is unknown. Fatty liver disease (steatosis) is associated with markers of UPR activation and robust UPR induction can cause steatosis; however, in other cases, UPR activation can protect against this disease. By assessing the magnitude of activation of UPR sensors and target genes in the liver of zebrafish larvae exposed to three commonly used ER stressors (tunicamycin, thapsigargin and Brefeldin A), we have identified distinct combinations of UPR sensors and targets (i.e. subclasses) activated by each stressor. We found that only the UPR subclass characterized by maximal induction of UPR target genes, which we term a stressed-UPR, induced steatosis. Principal component analysis demonstrated a significant positive association between UPR target gene induction and steatosis. The same principal component analysis showed significant correlation with steatosis in samples from patients with fatty liver disease. We demonstrate that an adaptive UPR induced by a short exposure to thapsigargin prior to challenging with tunicamycin reduced both the induction of a stressed UPR and steatosis incidence. We conclude that a stressed UPR causes steatosis and an adaptive UPR prevents it, demonstrating that this pathway plays dichotomous roles in fatty liver disease.