Neuroactivity screening of botanical extracts using microelectrode array (MEA) recordings

Toxicity testing of botanicals is challenging because of their chemical complexity and variability. Since botanicals may affect many different modes of action involved in neuronal function, we used microelectrode array (MEA) recordings of primary rat cortical cultures to screen 16 different botanical extracts for their effects on cell viability and neuronal network function in vitro. Our results demonstrate that extract materials (50 μg/mL) derived from goldenseal, milk thistle, tripterygium, and yohimbe decrease mitochondrial activity following 7 days exposure, indicative of cytotoxicity. Importantly, most botanical extracts alter neuronal network function following acute exposure. Extract materials (50 μg/mL) derived from aristolochia, ephedra, green tea, milk thistle, tripterygium, and usnea inhibit neuronal activity. Extracts of kava, kratom and yohimbe are particularly potent and induce a profound inhibition of neuronal activity at the low dose of 5 μg/mL. Extracts of blue cohosh, goldenseal and oleander cause intensification of the bursts. Aconite extract (5 μg/mL) evokes a clear hyperexcitation with a marked increase in the number of spikes and (network) bursts. The distinct activity patterns suggest that botanical extracts have diverse modes of action. Our combined data also highlight the applicability of MEA recordings for hazard identification and potency ranking of botanicals

New Approach Methodologies for the Endocrine Activity Toolbox: Environmental Assessment for Fish and Amphibians

Multiple in vivo test guidelines focusing on the estrogen, androgen, thyroid, and steroidogenesis pathways have been developed and validated for mammals, amphibians, or fish. However, these tests are resource-intensive and often use a large number of laboratory animals. Developing alternatives for in vivo tests is consistent with the replacement, reduction, and refinement principles for animal welfare considerations, which are supported by increasing mandates to move toward an “animal-free” testing paradigm worldwide. New approach methodologies (NAMs) hold great promise to identify molecular, cellular, and tissue changes that can be used to predict effects reliably and more efficiently at the individual level (and potentially on populations) while reducing the number of animals used in (eco)toxicological testing for endocrine disruption. In a collaborative effort, experts from government, academia, and industry met in 2020 to discuss the current challenges of testing for endocrine activity assessment for fish and amphibians. Continuing this cross-sector initiative, our review focuses on the current state of the science regarding the use of NAMs to identify chemical-induced endocrine effects. The present study highlights the challenges of using NAMs for safety assessment and what work is needed to reduce their uncertainties and increase their acceptance in regulatory processes. We have reviewed the current NAMs available for endocrine activity assessment including in silico, in vitro, and eleutheroembryo models. New approach methodologies can be integrated as part of a weight-of-evidence approach for hazard or risk assessment using the adverse outcome pathway framework. The development and utilization of NAMs not only allows for replacement, reduction, and refinement of animal testing but can also provide robust and fit-for-purpose methods to identify chemicals acting via endocrine mechanisms.publishedVersio

Comparative Analysis of Microfluidics Thrombus Formation in Multiple Genetically Modified Mice: Link to Thrombosis and Hemostasis

Genetically modified mice are indispensable for establishing the roles of platelets in arterial thrombosis and hemostasis. Microfluidics assays using anticoagulated whole blood are commonly used as integrative proxy tests for platelet function in mice. In the present study, we quantified the changes in collagen-dependent thrombus formation for 38 different strains of (genetically) modified mice, all measured with the same microfluidics chamber. The mice included were deficient in platelet receptors, protein kinases or phosphatases, small GTPases or other signaling or scaffold proteins. By standardized re-analysis of high-resolution microscopic images, detailed information was obtained on altered platelet adhesion, aggregation and/or activation. For a subset of 11 mouse strains, these platelet functions were further evaluated in rhodocytin- and laminin-dependent thrombus formation, thus allowing a comparison of glycoprotein VI (GPVI), C-type lectin-like receptor 2 (CLEC2) and integrin alpha(6)beta(1) pathways. High homogeneity was found between wild-type mice datasets concerning adhesion and aggregation parameters. Quantitative comparison for the 38 modified mouse strains resulted in a matrix visualizing the impact of the respective (genetic) deficiency on thrombus formation with detailed insight into the type and extent of altered thrombus signatures. Network analysis revealed strong clusters of genes involved in GPVI signaling and Ca2+ homeostasis. The majority of mice demonstrating an antithrombotic phenotype in vivo displayed with a larger or smaller reduction in multi-parameter analysis of collagen-dependent thrombus formation in vitro. Remarkably, in only approximately half of the mouse strains that displayed reduced arterial thrombosis in vivo, this was accompanied by impaired hemostasis. This was also reflected by comparing in vitro thrombus formation (by microfluidics) with alterations in in vivo bleeding time. In conclusion, the presently developed multi-parameter analysis of thrombus formation using microfluidics can be used to: (i) determine the severity of platelet abnormalities;(ii) distinguish between altered platelet adhesion, aggregation and activation;and (iii) elucidate both collagen and non-collagen dependent alterations of thrombus formation. This approach may thereby aid in the better understanding and better assessment of genetic variation that affect in vivo arterial thrombosis and hemostasis

1993-94 Progress Report

The 1993 edition of the Progress Reports was prepared for the Maine Wild Blueberry Commission and the University of Maine Wild Blueberry Advisory Committee by researchers at the University of Maine, Orono. Projects in this report include: 1. Effects of irrigation on lowbush blueberry yield and quality 2. The Economics of investigating irrigation for lowbush blueberries 3. Phosphorus dose/response curve 4. Winter injury protection by potassium 5. Multiple cropping of wild stands 6. Effect of Boron and Calcium on lowbush blueberry fruit set and yield 7. Comparison of N, NP, and NPK fertilizers to correct nitrogen and phosphorus deficiency 8. Determination of pesticide residue levels in freshly harvested and processed lowbush blueberries 9. Effects of calcium salts and citric acid on the quality of canned lowbush blueberries 10. Investigation of preprocess changes (chemical, microbiological, and/or physical) that can lead to the development of a simple and inexpensive method to measure preprocess berry spoilage 11. The effect of fertilization and irrigation in blueberry fruit quality 12. Pollination Ecology of lowbush blueberry in Maine 13. Current importance of insects in lowbush blueberry fields 14. Application of heat as a method of controlling secondary pest insects on lowbush blueberry: a feasibility study 15. Control of blueberry maggot 16. Control of secondary blueberry pest insects 17. Biology and action thresholds of secondary blueberry pest insects 18. Cold-hardiness of native lowbush blueberry 19. Design, fabrication, and testing of an experimental sterilizer for blueberry fields 20. Canned Product Quality--Heat-resistant molds 21. Sanitation for disease control 22. Evaluation of Velpar® impregnated DAP and Pronone® for weed control 23. Evaluation of postemergence applications of tribenuron methyl for bunchberry control 24. Evaluation of postemergence applications of a tank mix of tribenuron methyl and hexazinone for bunchberry control 25. Thresholds of dogbane and bracken fem by mechanical and chemical control in lowbush blueberry fields 26. Effect of time of application of clopyralid for control of vetch and effect on flowering in lowbush blueberries 27. Effect of time of fall pruning on growth and productivity of blueberries and evaluation of infrared burner to prune blueberries 28. Evaluation of infrared burner for selective seedling weed control 29. Evaluation of pressurized rope wick Wick Master wiper for treating weeds growing above lowbush blueberries 30. Blueberry Extension Education Program Base 31. Blueberry ICM program for Hancock County 32. Composting blueberry processing waste 33. Hexazinone ground water survey 34. Investigations of Lowbush Blueberry Fruit bud Cold-hardiness 35. Design, Fabrication, and Testing of an Experimental Sterilizer for Blueberry Field

Cancer Yield of Mammography, MR, and US in High-Risk Women: Prospective Multi-Institution Breast Cancer Screening Study

PURPOSE: To prospectively determine cancer yield, callback and biopsy rates, and positive predictive value (PPV) of mammography, magnetic resonance (MR) imaging, and ultrasonography (US) in women at high risk for breast cancer. MATERIALS AND METHODS: The study was approved by the institutional review board and was HIPAA compliant, and informed consent was obtained. We conducted a prospective pilot study of screening mammography, MR, and US in asymptomatic women 25 years of age or older who were genetically at high risk, defined as BRCA1/BRCA2 carriers or with at least a 20% probability of carrying a BRCA1/BRCA2 mutation. All imaging modalities were performed within 90 days of each other. Data were analyzed by using exact confidence intervals (CIs) and the McNemar test. RESULTS: A total of 195 women were enrolled in this study over a 6-month period, and 171 completed all study examinations (mammography, US, and MR). Average age of the 171 participants was 46 years +/- 10.2 (standard deviation). Sixteen biopsies were performed and six cancers were detected, for an overall 3.5% cancer yield. MR enabled detection of all six cancers; mammography, two; and US, one. The diagnostic yields for each test were 3.5% for MR, 0.6% for US, and 1.2% for mammography. MR, US, and mammography findings prompted biopsy in 8.2%, 2.3%, and 2.3% of patients, respectively. None of the pairwise comparisons were statistically significant. The PPV of biopsies performed as a result of MR was 43%. CONCLUSION: Screening MR imaging had a higher biopsy rate but helped detect more cancers than either mammography or US. US had the highest false-negative rate compared with mammography and MR, enabling detection of only one in six cancers in high-risk women

Stress sensitization and adolescent depressive severity as a function of childhood adversity: A link to anxiety disorders

The goal of the present study was to determine whether exposure to adversity in childhood contributes to a differential threshold at which stressful life events provoke depressive reactions in adolescence. In addition, to address empirical and conceptual questions about stress effects, the moderating effect of anxiety disorder history was also explored. This examination was conducted in a sample of 816 children of depressed and nondepressed mothers, who were followed from birth to age 15. Information on adversities experienced in childhood was collected both from mothers during the first five years of their youth's life and from the youths themselves at age 15, and included information on the mother's relationship with her partner, maternal psychopathology, as well as youth-reported abuse. Results indicated that youths with both greater exposure to adversity in childhood and a history of an anxiety disorder displayed increased depressive severity following low levels of episodic stress compared to youths with only one or neither of these risk factors. The results are speculated to reflect the possibility that early anxiety disorders associated with exposure to adversity in childhood may be a marker of dysregulated stress responses, and may help to account for the comorbidity of depression and anxiety in some individuals

Female Chromosome X Mosaicism is Age-Related and Preferentially Affects the Inactivated X Chromosome

To investigate large structural clonal mosaicism of chromosome X, we analysed the SNP microarray intensity data of 38,303 women from cancer genome-wide association studies (20,878 cases and 17,425 controls) and detected 124 mosaic X events 4 2 Mb in 97 (0.25%) women. Here we show rates for X-chromosome mosaicism are four times higher than mean autosomal rates; X mosaic events more often include the entire chromosome and participants with X events more likely harbour autosomal mosaic events. X mosaicism frequency increases with age (0.11% in 50-year olds; 0.45% in 75-year olds), as reported for Y and autosomes. Methylation array analyses of 33 women with X mosaicism indicate events preferentially involve the inactive X chromosome. Our results provide further evidence that the sex chromosomes undergo mosaic events more frequently than autosomes, which could have implications for understanding the underlying mechanisms of mosaic events and their possible contribution to risk for chronic diseases

JAK-STAT and AKT pathway-coupled genes in erythroid progenitor cells through ontogeny

Background: It has been reported that the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway regulates erythropoietin (EPO)-induced survival, proliferation, and maturation of early erythroid progenitors. Erythroid cell proliferation and survival have also been related to activation of the JAK-STAT pathway. The goal of this study was to observe the function of EPO activation of JAK-STAT and PI3K/AKT pathways in the development of erythroid progenitors from hematopoietic CD34(+) progenitor cells, as well as to distinguish early EPO target genes in human erythroid progenitors during ontogeny. Methods: Hematopoietic CD34(+) progenitor cells, isolated from fetal and adult hematopoietic tissues, were differentiated into erythroid progenitor cells. We have used microarray analysis to examine JAK-STAT and PI3K/AKT related genes, as well as broad gene expression modulation in these human erythroid progenitor cells. Results: In microarray studies, a total of 1755 genes were expressed in fetal liver, 3844 in cord blood, 1770 in adult bone marrow, and 1325 genes in peripheral blood-derived erythroid progenitor cells. The erythroid progenitor cells shared 1011 common genes. Using the Ingenuity Pathways Analysis software, we evaluated the network pathways of genes linked to hematological system development, cellular growth and proliferation. The KITLG, EPO, GATA1, PIM1 and STAT3 genes represent the major connection points in the hematological system development linked genes. Some JAK-STAT signaling pathway-linked genes were steadily upregulated throughout ontogeny (PIM1, SOCS2, MYC, PTPN11), while others were downregulated (PTPN6, PIAS, SPRED2). In addition, some JAK-STAT pathway related genes are differentially expressed only in some stages of ontogeny (STATs, GRB2, CREBB). Beside the continuously upregulated (AKT1, PPP2CA, CHUK, NFKB1) and downregulated (FOXO1, PDPK1, PIK3CG) genes in the PI3K-AKT signaling pathway, we also observed intermittently regulated gene expression (NFKBIA, YWHAH). Conclusions: This broad overview of gene expression in erythropoiesis revealed transcription factors differentially expressed in some stages of ontogenesis. Finally, our results show that EPO-mediated proliferation and survival of erythroid progenitors occurs mainly through modulation of JAK-STAT pathway associated STATs, GRB2 and PIK3 genes, as well as AKT pathway-coupled NFKBIA and YWHAH genes

MRI detection of distinct incidental cancer in women with primary breast cancer studied

Background: Prior single institution studies suggest MRI may improve the assessment of the extent of cancer within the breast, and thus reduce the risk of leaving macroscopic disease in the breast following breast conservation therapy. We report on the rate of MRI and mammography detection of foci of distinct incidental cancer in a prospective, multi center trial involving 426 women with confirmed breast cancer at 15 institutions in the US, Canada, and Germany. Methods: Women underwent mammography and MRI prior to biopsy of the suspicious index lesion. Additional incidental lesions (IL) greater than 2 cm from the index lesion that were detected by mammography and MRI were noted and characterized. Biopsy recommendations were associated with ILs given an assessment of suspicious or highly suspicous (BiRads 4 and 5). These assessments were considered a positive test. Results: MRI had a significantly higher yield of confirmed cancer ILs than mammography (0.18 (95%CI: 0.142-0.214) for MRI versus 0.072 (95%CI: 0.050-0.100) for mammography). The cancer ILs detected by MRI alone appeared to be similar to those detected by mammography with respect to size and histology. The percentage of biopsies of ILs that resulted in a cancer diagnosis was similar between Philadelphia, PA 19104, Fax: 215-662-7238, 215-662-301