Effects of anticholinergics and cholinergic enhancers on spatial learning and locomotion in mice

To investigate the cholinergic system’s role In learning and memory, I. utilized the Morris water maze (MWM) task, which tests spatial ability. Subjects must locate a submerged, hidden platform using the various distal cues around the room to orient themselves in space to find the escape platform. The cholinergic system regulates spatial learning and memory necessary for tasks such as the MWM and also plays a role in locomotor activity. I performed a pilot study to demonstrate that outbred strains of mice perform the MWM task effectively. I then used an outbred strain of mice, Swiss Webster, in the MWM to test the effects of a purported cholinergic enhancer and standard anticholinergics on spatial memory and on locomotor activity. Anticholinergics hinder an animal’s ability to perform spatial learning activities by blocking muscarinic acetylcholine (ACh) receptors In the neural synapses. In Experiment One, I tested the effects of the anticholinergic. Scopolamine (Scop), thereby inhibiting effects of ACh in the neural synapses. To attempt to reverse the effects of Scop, we used Schlsandrol A (SchisA), a natural acetylcholinesterase inhibitor. The drug Is thought to Improve memory formation by inhibiting the breakdown of ACh In the synapses, which increases ACh’s effect time for synaptic transmission. I found that SchisA did not reverse memory deficits Induced by Scop, In fact, the combined drug treatment group did worse than all other groups. In addition, Scop did not have as dramatic deficits on learning as expected; therefore, in Experiment Two, I tested the effects of Scop at a higher dosage along with another anticholinergic, Atropine Sulfate (AS), in the MWM. Both anticholinergics Induced learning and memory impairments; however, they Induced opposite locomotion effects with Scop Increasing and AS decreasing locomotion. Based on these results, I do not recommend further research into the possibility of SchlsA being a treatment option for ailments involving reduced cholinergic activity, such as Alzheimer’s disease. Additionally, the higher dosage of Scop should be used in studies where a learning deficit is desired, while the dosage of AS I used should be avoided In mice due to its substantial peripheral effects

The Effects of Apigenin on Cell Proliferation and Apoptosis in Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a WHO grade IV brain tumor. These tumors are highly proliferative, infiltrative, necrotic, angiogenic, and resistant to apoptosis. One major characteristic of GBM is the overexpression of epidermal growth factor receptor (EGFR), which leads to cell growth and proliferation when activated. GBM is very difficult to treat due to its location, heterogeneity, and invasiveness; an effective treatment is therefore needed. The use of flavonoids, which are natural compounds found in many fruits and vegetables, has been studied in the treatment of many different tumor types. Apigenin is a specific flavonoid that has previously been shown to have antitumor activity in a number of cancer cells. Our study set out to investigate the molecular effects of apigenin treatment on glioblastoma cell proliferation and viability using the trypan blue exclusion assay, MTT assay, and an LDH assay. In addition, Western blot analyses were utilized out to determine the signaling pathways through which apigenin treatment exerts its effects on cell proliferation and apoptosis. Finally, hoechst-propidium iodide staining and flow cytometry were used to examine the extent of apoptosis and the cell cycle context of these effects. Our results show that apigenin reduces cell viability and proliferation in a dose and time dependent manner while increasing cytotoxicity in GBM cells. Additionally, apigenin inhibits the EGFR mediated phosphorylation in the presence of EGF treatment of AKT, mTOR, and s6k resulting in decreased cell survival, growth and proliferation. It also inhibits the MAPK pathways in one cell line thereby reducing cell growth and proliferation. It also inhibits the anti-apoptotic effects of BCL-XL and increases PARP cleavage, which leads to increased apoptosis. Finally, apigenin induced cycle arrest at the G2M checkpoint, meaning that apoptosis primarily occurred at the DNA repair checkpoint in the cell cycle. In conclusion, apigenin has demonstrated some in vitro biological effects on glioblastoma cell lines that show promises in limiting the growth, proliferation and survival of these cell lines. Future research should look to identify means through which apigenin can be administered in clinically significant concentrations to the brain

Natural Products as Therapeutic Agents in Cancer Treatment

Cancer accounts for 25% of deaths in the United States, and brain tumors greatly contribute to this percentage. However, relative to other types of cancers, brain tumors prove difficult to treat because they are heterogeneous, highly proliferative, highly invasive, and resistant to the traditional cancer treatments of chemotherapy and radiotherapy. Past studies have shown that flavonoids and curcuminoids, two classes of compounds derived from natural sources, are effective in inhibiting the development and metastasis of breast and lung cancer cells. Research has also indicated that these compounds have potential for treating brain tumors. The purpose of this research is to further explore the potential of flavonoids as therapeutic options for the treatment of brain tumors. Specifically, flavonoids’ effect on cell proliferation, cell death, and tumor invasion will be studied. Another objective of this study is to identify the signaling mechanism by which flavonoids mediate their therapeutic effects on brain tumor cell lines. Three human brain tumor cell lines (U-1242, U-251, and U-87) will be studied. They will be treated with various flavonoids at increasing concentrations (10, 20, 40, and 80 µM). Cells will be counted following the trypan blue staining protocol. MTT assays and Western blot analyses will be used to assess cell proliferation. Cell death will be assessed with flow analyses and Western blot analyses. Unpaired t-tests will be run to compare treated and control cells at a 95% confidence interval. If necessary, one-way ANOVA with multiple comparisons will be used to compare multiple treatment groups and a control at a 95% confidence interval, and the Tukey post-hoc test will be utilized if appropriate. All statistical tests will be run in IBM SPSS 21®

Natural Products as Therapeutic Agents in Cancer Treatment

Cancer accounts for 25% of deaths in the United States, and brain tumors greatly contribute to this percentage. However, relative to other types of cancers, brain tumors prove difficult to treat because they are heterogeneous, highly proliferative, highly invasive, and resistant to the traditional cancer treatments of chemotherapy and radiotherapy. Past studies have shown that flavonoids and curcuminoids, two classes of compounds derived from natural sources, are effective in inhibiting the development and metastasis of breast and lung cancer cells. Research has also indicated that these compounds have potential for treating brain tumors. The purpose of this research is to further explore the potential of flavonoids as therapeutic options for the treatment of brain tumors. Specifically, flavonoids’ effect on cell proliferation, cell death, and tumor invasion will be studied. Another objective of this study is to identify the signaling mechanism by which flavonoids mediate their therapeutic effects on brain tumor cell lines. Three human brain tumor cell lines (U-1242, U-251, and U-87) will be studied. They will be treated with various flavonoids at increasing concentrations (10, 20, 40, and 80 µM). Cells will be counted following the trypan blue staining protocol. MTT assays and Western blot analyses will be used to assess cell proliferation. Cell death will be assessed with flow analyses and Western blot analyses. Unpaired t-tests will be run to compare treated and control cells at a 95% confidence interval. If necessary, one-way ANOVA with multiple comparisons will be used to compare multiple treatment groups and a control at a 95% confidence interval, and the Tukey post-hoc test will be utilized if appropriate. All statistical tests will be run in IBM SPSS 21®

The Antiproliferative and Apoptotic Effects of Apigenin on Glioblastoma Cells

OBJECTIVES: Glioblastoma (GBM) is highly proliferative, infiltrative, malignant and the most deadly form of brain tumour. The epidermal growth factor receptor (EGFR) is overexpressed, amplified and mutated in GBM and has been shown to play key and important roles in the proliferation, growth and survival of this tumour. The goal of our study was to investigate the antiproliferative, apoptotic and molecular effects of apigenin in GBM. METHODS: Proliferation and viability tests were carried out using the trypan blue exclusion, MTT and lactate dehydrogenase (LDH) assays. Flow cytometry was used to examine the effects of apigenin on the cell cycle check-points. In addition, we determined the effects of apigenin on EGFR-mediated signalling pathways by Western blot analyses. KEY FINDINGS: Our results showed that apigenin reduced cell viability and proliferation in a dose- and time-dependent manner while increasing cytotoxicity in GBM cells. Treatment with apigenin-induced is poly ADP-ribose polymerase (PARP) cleavage and caused cell cycle arrest at the G2M checkpoint. Furthermore, our data revealed that apigenin inhibited EGFR-mediated phosphorylation of mitogen-activated protein kinase (MAPK), AKT and mammalian target of rapamycin (mTOR) signalling pathways and attenuated the expression of Bcl-xL. CONCLUSION: Our results demonstrated that apigenin has potent inhibitory effects on pathways involved in GBM proliferation and survival and could potentially be used as a therapeutic agent for GBM

Development of a High-Throughput Screening Assay to Identify Inhibitors of the Lipid Kinase PIP5K1C

Phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) regulate a variety of cellular processes including signaling through G protein-coupled receptors (GPCRs), endocytosis, exocytosis, and cell migration. These lipid kinases synthesize phosphatidylinositol 4,5-bisphosphate (PIP2) from phosphatidylinositol 4-phosphate [PI(4)P]. Since small molecule inhibitors of these lipid kinases did not exist, molecular and genetic approaches were predominantly used to study PIP5K1 regulation of these cellular processes. Moreover, standard radioisotope-based lipid kinase assays cannot be easily adapted for high-throughput screening. Here, we report a novel high-throughput microfluidic mobility shift assay to identify inhibitors of PIP5K1C. This assay utilizes fluorescently labeled phosphatidylinositol 4-phosphate as the substrate and recombinant human PIP5K1C. Our assay exhibited high reproducibility, had a calculated ATP Km of 15 µM, performed with z’ values >0.7, and was used to screen a kinase-focused library of ~4,700 compounds. From this screen, we identified several potent inhibitors of PIP5K1C, including UNC3230, a compound that we recently found can reduce nociceptive sensitization in animal models of chronic pain. This novel assay will allow continued drug discovery efforts for PIP5K1C and can be easily adapted to screen additional lipid kinases

Diagnosis and management in Rubinstein-Taybi syndrome:first international consensus statement

Rubinstein-Taybi syndrome (RTS) is an archetypical genetic syndrome that is characterised by intellectual disability, well-defined facial features, distal limb anomalies and atypical growth, among numerous other signs and symptoms. It is caused by variants in either of two genes (CREBBP, EP300) which encode for the proteins CBP and p300, which both have a function in transcription regulation and histone acetylation. As a group of international experts and national support groups dedicated to the syndrome, we realised that marked heterogeneity currently exists in clinical and molecular diagnostic approaches and care practices in various parts of the world. Here, we outline a series of recommendations that document the consensus of a group of international experts on clinical diagnostic criteria for types of RTS (RTS1: CREBBP; RTS2: EP300), molecular investigations, long-term management of various particular physical and behavioural issues and care planning. The recommendations as presented here will need to be evaluated for improvements to allow for continued optimisation of diagnostics and care.</p

Diagnosis and management in Rubinstein-Taybi syndrome:first international consensus statement

Rubinstein-Taybi syndrome (RTS) is an archetypical genetic syndrome that is characterised by intellectual disability, well-defined facial features, distal limb anomalies and atypical growth, among numerous other signs and symptoms. It is caused by variants in either of two genes (CREBBP, EP300) which encode for the proteins CBP and p300, which both have a function in transcription regulation and histone acetylation. As a group of international experts and national support groups dedicated to the syndrome, we realised that marked heterogeneity currently exists in clinical and molecular diagnostic approaches and care practices in various parts of the world. Here, we outline a series of recommendations that document the consensus of a group of international experts on clinical diagnostic criteria for types of RTS (RTS1: CREBBP; RTS2: EP300), molecular investigations, long-term management of various particular physical and behavioural issues and care planning. The recommendations as presented here will need to be evaluated for improvements to allow for continued optimisation of diagnostics and care.</p

The daily association between affect and alcohol use: a meta-analysis of individual participant data

Influential psychological theories hypothesize that people consume alcohol in response to the experience of both negative and positive emotions. Despite two decades of daily diary and ecological momentary assessment research, it remains unclear whether people consume more alcohol on days they experience higher negative and positive affect in everyday life. In this preregistered meta-analysis, we synthesized the evidence for these daily associations between affect and alcohol use. We included individual participant data from 69 studies (N = 12,394), which used daily and momentary surveys to assess affect and the number of alcoholic drinks consumed. Results indicate that people are not more likely to drink on days they experience high negative affect, but are more likely to drink and drink heavily on days high in positive affect. People self-reporting a motivational tendency to drink-to-cope and drink-to-enhance consumed more alcohol, but not on days they experienced higher negative and positive affect. Results were robust across different operationalizations of affect, study designs, study populations, and individual characteristics. These findings challenge the long-held belief that people drink more alcohol following increases in negative affect. Integrating these findings under different theoretical models and limitations of this field of research, we collectively propose an agenda for future research to explore open questions surrounding affect and alcohol use.The present study was funded by the Canadian Institutes of Health Research Grant MOP-115104 (Roisin M. O’Connor), Canadian Institutes of Health Research Grant MSH-122803 (Roisin M. O’Connor), John A. Hartford Foundation Grant (Paul Sacco), Loyola University Chicago Research Support Grant (Tracy De Hart), National Institute for Occupational Safety and Health Grant T03OH008435 (Cynthia Mohr), National Institutes of Health (NIH) Grant F31AA023447 (Ryan W. Carpenter), NIH Grant R01AA025936 (Kasey G. Creswell), NIH Grant R01AA025969 (Catharine E. Fairbairn), NIH Grant R21AA024156 (Anne M. Fairlie), NIH Grant F31AA024372 (Fallon Goodman), NIH Grant R01DA047247 (Kevin M. King), NIH Grant K01AA026854 (Ashley N. Linden-Carmichael), NIH Grant K01AA022938 (Jennifer E. Merrill), NIH Grant K23AA024808 (Hayley Treloar Padovano), NIH Grant P60AA11998 (Timothy Trull), NIH Grant MH69472 (Timothy Trull), NIH Grant K01DA035153 (Nisha Gottfredson), NIH Grant P50DA039838 (Ashley N. Linden-Carmichael), NIH Grant K01DA047417 (David M. Lydon-Staley), NIH Grant T32DA037183 (M. Kushner), NIH Grant R21DA038163 (A. Moore), NIH Grant K12DA000167 (M. Potenza, Stephanie S. O’Malley), NIH Grant R01AA025451 (Bruce Bartholow, Thomas M. Piasecki), NIH Grant P50AA03510 (V. Hesselbrock), NIH Grant K01AA13938 (Kristina M. Jackson), NIH Grant K02AA028832 (Kevin M. King), NIH Grant T32AA007455 (M. Larimer), NIH Grant R01AA025037 (Christine M. Lee, M. Patrick), NIH Grant R01AA025611 (Melissa Lewis), NIH Grant R01AA007850 (Robert Miranda), NIH Grant R21AA017273 (Robert Miranda), NIH Grant R03AA014598 (Cynthia Mohr), NIH Grant R29AA09917 (Cynthia Mohr), NIH Grant T32AA07290 (Cynthia Mohr), NIH Grant P01AA019072 (P. Monti), NIH Grant R01AA015553 (J. Morgenstern), NIH Grant R01AA020077 (J. Morgenstern), NIH Grant R21AA017135 (J. Morgenstern), NIH Grant R01AA016621 (Stephanie S. O’Malley), NIH Grant K99AA029459 (Marilyn Piccirillo), NIH Grant F31AA022227 (Nichole Scaglione), NIH Grant R21AA018336 (Katie Witkiewitz), Portuguese State Budget Foundation for Science and Technology Grant UIDB/PSI/01662/2020 (Teresa Freire), University of Washington Population Health COVID-19 Rapid Response Grant (J. Kanter, Adam M. Kuczynski), U.S. Department of Defense Grant W81XWH-13-2-0020 (Cynthia Mohr), SANPSY Laboratory Core Support Grant CNRS USR 3413 (Marc Auriacombe), Social Sciences and Humanities Research Council of Canada Grant (N. Galambos), and Social Sciences and Humanities Research Council of Canada Grant (Andrea L. Howard)