Calcium handling genes are regulated by promoter DNA methylation in colorectal cancer cells.

Calcium signaling regulates various cellular processes, including proliferation and cell death. DNA methylation of gene promoters is an epigenetic modification that facilitates transcriptional suppression. Disruption of calcium homeostasis and DNA methylation in cancer are each linked to tumor development and progression. However, the possible connection between these two processes has not been thoroughly studied. Therefore, we measured the expression of six gene families involved in calcium regulation (ATP2A, ITPR, ORAI, RyR, STIM, and TRPC) in a colorectal cancer cell model, HCT116, with either genetic (Double Knock-out/DKO) or pharmacological (5-aza-2'-deoxycytidine/DAC) inhibition of DNA methyltransferases. Fourteen of the 20 examined calcium handling genes were expressed at higher levels in DKO cells as compared to HCT116. Expression of five genes was increased in HCT116 cells treated with DAC, three matching DKO. Due to a unique expression pattern of the three ATP2A genes in our model, encoding the Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase (SERCA) pumps, we chose to evaluate the methylation status of these genes, protein expression, and potential associated physiological effects, using the SERCA inhibitor thapsigarin (TG). We observed an expected pattern of promoter methylation coinciding with reduced expression and vice versa. This differential mRNA expression was associated with altered SERCA3 protein expression and cytosolic calcium levels with TG exposure. As a result, DKO cells displayed less TG-induced cytotoxicity, as compared to HCT116 cells. Overall, it is likely that at least several calcium regulatory genes are transcriptionally regulated by DNA methylation, and this may play a role in tumorigenesis through altering apoptosis in cancer.

Development and assessment of a nationwide, cross-discipline women faculty mentoring program.

INTRODUCTION: Professional health care associations present a unique opportunity for formal mentorship programs, with membership often spanning a variety of experiences, professional ranks, and institutions. However, scarce literature describes the role of professional associations in the development and assessment of mentoring programs. This paper describes development of the American Association of Colleges of Pharmacy Women Faculty Special Interest Group (SIG) mentoring program and characterizes the impact of the program. METHODS: The task force collaboratively developed the mission, vision, and structure of the mentoring program, posted the program description on the SIG's electronic forum, and called for mentors and mentees via an online survey asking for matching preferences. The task force reviewed responses and designated matches. Participants were emailed match information and a guidance document. The program was assessed at three, six, and 12 months via electronic survey. RESULTS: The program matched 43 mentors with 77 mentees, with each mentor assigned one to three mentees. At the three- and six-month assessments, 89% and 87% of respondents, respectively, indicated they had met with their mentor/mentee. At the 12-month assessment, 86% of mentor respondents and 80% of mentee respondents stated the mentoring program met their needs/expectations. Career goal development, work/life integration, and difficult work situations were the most frequently discussed topics. Most participants stated they would continue to serve as a mentor/mentee in a future cycle and recommend other faculty members participate. CONCLUSIONS: Results from three-, six-, and 12-month assessments indicated a positive impact of developing a nationwide, organizational, cross-discipline mentoring program.

Student and faculty perceptions of integrated therapeutics courses in a doctor of pharmacy program.

INTRODUCTION: There has been a movement in clinical education towards integrating foundational and applied course content. It remains unclear what best practices are for integration or how meaningful integration is for improving learning. We assessed the impact and perceptions of different degrees of integration in Integrated Pharmacy Care and Patient Management (IPC) courses. METHODS: Students and faculty of one four-year doctor of pharmacy program were asked to complete surveys upon conclusion of the last of twelve integrated courses. Three degrees of integration (first degree, second degree, complete Integration) were defined for respondents. The student questionnaire included items on perceived value of integrated education as well as used and preferred levels of integration throughout the course series. Perceived estimated integration levels were then correlated with mean grade point averages (GPAs) for the courses. The faculty survey assessed experience with, and perceptions of, integrated teaching. RESULTS: Sixty-five students (100% response) and 10 faculty (50% response) completed the surveys. Students preferred complete integration for all IPC courses and indicated that they were better able to retain and understand information with higher levels of integration. Supporting this, mean course GPAs positively correlated with estimated levels of integration. Faculty remained neutral on whether integration helps students to better understand course material and highlighted barriers to implementing higher levels of integration, including lack of time. CONCLUSIONS: Our results suggest that a team approach to complete integration is most beneficial for pharmacy students. Faculty may require additional institutional support to accomplish such integration.

Development of a laboratory-based pharmacogenomics independent study and advanced pharmacy practice experience: Connecting basic science to clinical application.

BACKGROUND AND PURPOSE: The role of pharmacists in pharmacogenomics (PGx) use clinically is expanding, leading to increased pharmacy education requirements. Current reports indicate that PGx is primarily taught through didactic courses, indicating a need for applied coursework in pharmacy curricula, including laboratory exercises and clinical experiences. Such courses are instrumental in helping students connect the science of PGx to patient care. EDUCATIONAL ACTIVITY AND SETTING: An advanced PGx independent study and a similar advanced pharmacy practice experience (APPE) were developed. These courses included personal genetic testing, raw genetic sequence data analysis, and wet-laboratory genetic testing. The APPE included sessions with clinical pharmacists who use PGx and a genetic counselor, as well as a visit to a genetic reference laboratory. A pre-/post-examination and survey were used to measure the courses' effectiveness and student perceptions of their abilities, PGx, and course components. For this pilot study one student per course was evaluated. FINDINGS: Each student completed all components of the courses successfully, supporting the feasibility of their implementation. Examination scores increased for both students with improvement in knowledge from basic genetics to clinical application. Both students also had a more positive perception of PGx after the courses and valued the various course components. SUMMARY: Through this unique course format, pharmacy students developed expertise in understanding and implementing PGx which allowed them to gain skills that go beyond an introductory course. Our experience may provide guidance to other pharmacy programs in adding more applied PGx education to their curricula.

Pharmacogenomics Education Improves Pharmacy Student Perceptions of Their Abilities and Roles in Its Use.

Objective. To assess whether a required first-year course, Principles in Genetics and Pharmacogenomics, and integration into subsequent courses affected pharmacy students' perceptions of pharmacogenomics. Methods. A survey was distributed to Professional Year (PY) 1 students during the first and last weeks of the course from 2014 to 2016. A follow-up survey was distributed to PY2, PY3, and PY4 students. Results. Respondents consistently agreed that pharmacogenomics is clinically relevant. After the course, PY1 students are more comfortable in their knowledge and role in the application of pharmacogenomics. Although their comfort reverts to some degree, PY2-PY4 students believe that they should be able to apply pharmacogenomics clinically. Most PY2-PY4 students indicate that later courses review pharmacogenomics. Conclusion. A required course in genetics and pharmacogenomics can promote a perception that pharmacists should have knowledge of, and be involved in the use of genetic information clinically. Inclusion of pharmacogenomic concepts in subsequent curricular components may help in maintaining these perceptions.

Epigenetic Regulation via Altered Histone Acetylation Results in Suppression of Mast Cell Function and Mast Cell-Mediated Food Allergic Responses.

Mast cells are highly versatile cells that perform a variety of functions depending on the immune trigger, context of activation, and cytokine stimulus. Antigen-mediated mast cell responses are regulated by transcriptional processes that result in the induction of numerous genes contributing to mast cell function. Recently, we also showed that exposure to dietary agents with known epigenetic actions such as curcumin can suppress mast cell-mediated food allergy, suggesting that mast cell responses in vivo may be epigenetically regulated. To further assess the effects of epigenetic modifications on mast cell function, we examined the behavior of bone marrow-derived mast cells (BMMCs) in response to trichostatin A (TSA) treatment, a well-studied histone deacetylase inhibitor. IgE-mediated BMMC activation resulted in enhanced expression and secretion of IL-4, IL-6, TNF-α, and IL-13. In contrast, pretreatment with TSA resulted in altered cytokine secretion. This was accompanied by decreased expression of FcεRI and mast cell degranulation. Interestingly, exposure to non-IgE stimuli such as IL-33, was also affected by TSA treatment. Furthermore, continuous TSA exposure contributed to mast cell apoptosis and a decrease in survival. Further examination revealed an increase in I-κBα and a decrease in phospho-relA levels in TSA-treated BMMCs, suggesting that TSA alters transcriptional processes, resulting in enhancement of I-κBα transcription and decreased NF-κB activation. Lastly, treatment of wild-type mice with TSA in a model of ovalbumin-induced food allergy resulted in a significant attenuation in the development of food allergy symptoms including decreases in allergic diarrhea and mast cell activation. These data therefore suggest that the epigenetic regulation of mast cell activation during immune responses may occur via altered histone acetylation, and that exposure to dietary substances may induce epigenetic modifications that modulate mast cell function.

IL-10 Enhances IgE-Mediated Mast Cell Responses and Is Essential for the Development of Experimental Food Allergy in IL-10-Deficient Mice.

IL-10 is a key pleiotropic cytokine that can both promote and curb Th2-dependent allergic responses. In this study, we demonstrate a novel role for IL-10 in promoting mast cell expansion and the development of IgE-mediated food allergy. Oral OVA challenge in sensitized BALB/c mice resulted in a robust intestinal mast cell response accompanied by allergic diarrhea, mast cell activation, and a predominance of Th2 cytokines, including enhanced IL-10 expression. In contrast, the development of intestinal anaphylaxis, including diarrhea, mast cell activation, and Th2 cytokine production, was significantly attenuated in IL-10(-/-) mice compared with wild-type (WT) controls. IL-10 also directly promoted the expansion, survival, and activation of mast cells; increased FcεRI expression on mast cells; and enhanced the production of mast cell cytokines. IL-10(-/-) mast cells had reduced functional capacity, which could be restored by exogenous IL-10. Similarly, attenuated passive anaphylaxis in IL-10(-/-) mice could be restored by IL-10 administration. The adoptive transfer of WT mast cells restored allergic symptoms in IL-10(-/-) mice, suggesting that the attenuated phenotype observed in these animals is due to a deficiency in IL-10-responding mast cells. Lastly, transfer of WT CD4 T cells also restored allergic diarrhea and intestinal mast cell numbers in IL-10(-/-) mice, suggesting that the regulation of IL-10-mediated intestinal mast cell expansion is T cell dependent. Our observations demonstrate a critical role for IL-10 in driving mucosal mast cell expansion and activation, suggesting that, in its absence, mast cell function is impaired, leading to attenuated food allergy symptoms.

Curcumin Ingestion Inhibits Mastocytosis and Suppresses Intestinal Anaphylaxis in a Murine Model of Food Allergy.

IgE antibodies and mast cells play critical roles in the establishment of allergic responses to food antigens. Curcumin, the active ingredient of the curry spice turmeric, has anti-inflammatory properties, and thus may have the capacity to regulate Th2 cells and mucosal mast cell function during allergic responses. We assessed whether curcumin ingestion during oral allergen exposure can modulate the development of food allergy using a murine model of ovalbumin (OVA)-induced intestinal anaphylaxis. Herein, we demonstrate that frequent ingestion of curcumin during oral OVA exposure inhibits the development of mastocytosis and intestinal anaphylaxis in OVA-challenged allergic mice. Intragastric (i.g.) exposure to OVA in sensitized BALB/c mice induced a robust IgE-mediated response accompanied by enhanced OVA-IgE levels, intestinal mastocytosis, elevated serum mMCP-1, and acute diarrhea. In contrast, mice exposed to oral curcumin throughout the experimental regimen appeared to be normal and did not exhibit intense allergic diarrhea or a significant enhancement of OVA-IgE and intestinal mast cell expansion and activation. Furthermore, allergic diarrhea, mast cell activation and expansion, and Th2 responses were also suppressed in mice exposed to curcumin during the OVA-challenge phase alone, despite the presence of elevated levels of OVA-IgE, suggesting that curcumin may have a direct suppressive effect on intestinal mast cell activation and reverse food allergy symptoms in allergen-sensitized individuals. This was confirmed by observations that curcumin attenuated the expansion of both adoptively transferred bone marrow-derived mast cells (BMMCs), and inhibited their survival and activation during cell culture. Finally, the suppression of intestinal anaphylaxis by curcumin was directly linked with the inhibition of NF-κB activation in curcumin-treated allergic mice, and curcumin inhibited the phosphorylation of the p65 subunit of NF-κB in BMMCs. In summary, our data demonstrates a protective role for curcumin during allergic responses to food antigens, suggesting that frequent ingestion of this spice may modulate the outcome of disease in susceptible individuals.

Nanopores suggest a negligible influence of CpG methylation on nucleosome packaging and stability.

Nucleosomes are the fundamental repeating units of chromatin, and dynamic regulation of their positioning along DNA governs gene accessibility in eukaryotes. Although epigenetic factors have been shown to influence nucleosome structure and dynamics, the impact of DNA methylation on nucleosome packaging remains controversial. Further, all measurements to date have been carried out under zero-force conditions. In this paper, we present the first automated force measurements that probe the impact of CpG DNA methylation on nucleosome stability. In solid-state nanopore force spectroscopy, a nucleosomal DNA tail is captured into a pore and pulled on with a time-varying electrophoretic force until unraveling is detected. This is automatically repeated for hundreds of nucleosomes, yielding statistics of nucleosome lifetime vs electrophoretic force. The force geometry, which is similar to displacement forces exerted by DNA polymerases and helicases, reveals that nucleosome stability is sensitive to DNA sequence yet insensitive to CpG methylation. Our label-free method provides high-throughput data that favorably compares with other force spectroscopy experiments and is suitable for studying a variety of DNA-protein complexes.

Fast, label-free force spectroscopy of histone-DNA interactions in individual nucleosomes using nanopores.

Herein we report a novel approach for fast, label-free probing of DNA-histone interactions in individual nucleosomes. We use solid-state nanopores to unravel individual DNA/histone complexes for the first time and find that the unraveling time depends on the applied electrophoretic force, and our results are in line with previous studies that employ optical tweezers. Our approach for studying nucleosomal interactions can greatly accelerate the understanding of fundamental mechanisms by which transcription, replication, and repair processes in a cell are modulated through DNA-histone interactions, as well as in diagnosis of diseases with abnormal patterns of DNA and histone modifications.