Combined BRCA2 and MAGEC3 Expression Predict Outcome in Advanced Ovarian Cancers.

Like BRCA2, MAGEC3 is an ovarian cancer predisposition gene that has been shown to have prognostic significance in ovarian cancer patients. Despite the clinical significance of each gene, no studies have been conducted to assess the clinical significance of their combined expression. We therefore sought to determine the relationship between MAGEC3 and BRCA2 expression in ovarian cancer and their association with patient characteristics and outcomes. Immunohistochemical staining was quantitated on tumor microarrays of human tumor samples obtained from 357 patients with epithelial ovarian cancer to ascertain BRCA2 expression levels. In conjunction with our previously published MAGEC3 expression data, we observed a weak inverse correlation of MAGEC3 with BRCA2 expression (r = −0.15; p < 0.05) in cases with full-length BRCA2. Patients with optimal cytoreduction, loss of MAGEC3, and detectable BRCA2 expression had better overall (median OS: 127.9 vs. 65.3 months, p = 0.035) and progression-free (median PFS: 85.3 vs. 18.8 months, p = 0.002) survival compared to patients that were BRCA2 expressors with MAGEC3 normal levels. Our results suggest that combined expression of MAGEC3 and BRCA2 serves as a better predictor of prognosis than each marker alone.

Coproducing the Learning Environment: Lessons Learned from a Year of Near-Peer Teaching.

PURPOSE: Few medical schools offer electives with the goal of teaching medical students to be effective teachers prior to residency. We developed a novel year-long, longitudinal course, the Clinical Teaching Elective (CTE), that develops fourth-year medical students as student teachers within Clinical Skills (CS). APPROACH/METHODS: The elective was designed by Clinical Skills (CS) Course Directors and two fourth-year medical students (M4) as a longitudinal elective. The elective involves teaching in the Simulation Center where M4 student instructors teach first and second-year medical students. Each session, in addition to simulated patient case topics, emphasizes application of a key topic within medical education (ie clinical reasoning, reflective practice, dual process reasoning). DISCUSSION: Six "teaching takeaways" were crafted to summarize common themes experienced by near-peer medical student educators. Teaching is not about the destination, but rather the diagnostic journey.Students thrive when learning is co-produced.A little bit of praise goes a long way.You can't please every learner.When students struggle, there is more to teach than just the answer.Facilitating learner independent thinking promotes future autonomy. SIGNIFICANCE: A novel CTE for fourth-year medical students that emphasizes medical education pedagogy prepares students to serve as educators in residency. The CTE provides an opportunity for medical students to develop into effective clinical educators prior to residency. The focus of our elective on medical education pedagogy furthers medical student understanding of adult learning theory and fosters professional development in teaching clinical reasoning.

Well-woman care and HPV vaccination rates in women with Rett syndrome.

BACKGROUND: Lifespan has increased in individuals with Rett syndrome (RTT), but little is currently known about the provision of well-woman care to these individuals. OBJECTIVE: To collect data on well-woman examinations and human papillomavirus (HPV) vaccination rates in women with RTT to understand the current state of women's healthcare in individuals with RTT. METHODS: A retrospective cross-sectional chart review and prospective survey of 77 patients with Rett syndrome who were cared for at a single specialty clinic over five years was conducted to collect data on women's health examinations and HPV vaccination rates. RESULTS: The following percentages represent women with RTT who have met the recommendations of ACOG for well-woman examinations: breast examinations- 40.3%, pelvic examinations- 51.2%, mammograms- 75.0%, external genitourinary examinations -31.6%. Many of these women also had delayed exams. 22.9% of women who were eligible for the HPV vaccine have received it. CONCLUSIONS: Many women with RTT do not undergo well-woman examinations and HPV vaccinations as recommended by ACOG. Since these women are not usually sexually active, many guardians believe the HPV vaccine is unnecessary. However, like other women with disabilities, RTT females are at risk for sexual abuse and disparities in access to women's health services, so these topics should be discussed with caretakers.

Features of Menstruation and Menstruation Management in Individuals with Rett Syndrome.

STUDY OBJECTIVE: To describe features of menstruation, menstrual-related symptoms, and menstrual management in females with Rett syndrome (RTT) to help develop a clinical approach to these parameters in RTT. DESIGN: Retrospective cross-sectional chart review and prospective survey. SETTING: Cincinnati Children's Hospital Medical Center, Rett Syndrome and Related Spectrum Disorders Clinic. PARTICIPANTS: Females with RTT (12-55 years of age) and their caregivers. MAIN OUTCOME MEASURES: Descriptive data on features of menstruation and menstrual-related symptoms in individuals with Rett syndrome; prevalence, types, reason for use/discontinuation, and efficacy of hormonal treatment in females with RTT. RESULTS: Age at menarche, menstrual cycle length, and menstrual period length in females with RTT are comparable to those in typically developing females and females with other neurodevelopmental disabilities. Dysmenorrhea and emotional lability are common menstrual cycle-related changes among females with RTT; 22.1% of participants also reported catamenial seizures. Oral progestin, combined oral contraceptive pill, and depot-medroxyprogesterone acetate (DMPA) were effectively used to suppress or regulate menstruation and to manage menstrual-related symptoms. CONCLUSIONS: Characteristics of menstruation in females with RTT are comparable to those of typically developing females, with the exception of increase in catamenial seizure activity. Hormonal treatments are used for management of menstruation, dysmenorrhea, and seizures. Choice of hormonal treatment is influenced by bone health and immobility in females with RTT.

Evolution of Distinct Responses to Low NAD+ Stress by Rewiring the Sir2 Deacetylase Network in Yeasts.

Evolutionary adaptation increases the fitness of a species in its environment. It can occur through rewiring of gene regulatory networks, such that an organism responds appropriately to environmental changes. We investigated whether sirtuin deacetylases, which repress transcription and require NAD+ for activity, serve as transcriptional rewiring points that facilitate the evolution of potentially adaptive traits. If so, bringing genes under the control of sirtuins could enable organisms to mount appropriate responses to stresses that decrease NAD+ levels. To explore how the genomic targets of sirtuins shift over evolutionary time, we compared two yeast species, Saccharomyces cerevisiae and Kluyveromyces lactis, that display differences in cellular metabolism and life cycle timing in response to nutrient availability. We identified sirtuin-regulated genes through a combination of chromatin immunoprecipitation and RNA expression. In both species, regulated genes were associated with NAD+ homeostasis, mating, and sporulation, but the specific genes differed. In addition, regulated genes in K. lactis were associated with other processes, including utilization of nonglucose carbon sources, detoxification of arsenic, and production of the siderophore pulcherrimin. Consistent with the species-restricted regulation of these genes, sirtuin deletion affected relevant phenotypes in K. lactis but not S. cerevisiae Finally, sirtuin-regulated gene sets were depleted for broadly conserved genes, consistent with sirtuins regulating processes restricted to a few species. Taken together, these results are consistent with the notion that sirtuins serve as rewiring points that allow species to evolve distinct responses to low NAD+ stress.

Establishing a role for environmental toxicant exposure induced epigenetic remodeling in malignant transformation.

Humans are exposed to a wide variety of environmental exposures throughout their lifespan. These include both naturally occurring toxins and chemical toxicants like pesticides, herbicides, and industrial chemicals, many of which have been implicated as possible contributors to human disease susceptibility [1-3]. We, and others, have hypothesized that environmental exposures may cause adaptive epigenetic changes in regenerative cell populations and developing organisms, leading to abnormal gene expression and increased disease susceptibility later in life [3]. Common epigenetic changes include changes in miRNA expression, covalent histone modifications, and methylation of DNA. Importantly, due to their heritable nature, abnormal epigenetic modifications which occur within stem cells may be particularly deleterious. Abnormal epigenetic changes in regenerative cell linages can be passed onto a large population of daughter cells and can persist for long periods of time. It is well established that an accumulation of epigenetic changes can lead to many human diseases including cancer [4-6]. Subsequently, it is imperative that we increase our understanding of how common environmental toxins and toxicants can induce epigenetic changes, particularly in stem cell populations. In this review, we will discuss how common environmental exposures in the United States and around the world may lead to epigenetic changes and discuss potential links to human disease, including cancer.

MPP+ decreases store-operated calcium entry and TRPC1 expression in Mesenchymal Stem Cell derived dopaminergic neurons.

Parkinson's disease is a neurodegenerative disorder involving the progressive loss of dopaminergic neurons (DNs), with currently available therapeutics, such as L-Dopa, only able to relieve some symptoms. Stem cell replacement is an attractive therapeutic option for PD patients, and DNs derived by differentiating patient specific stem cells under defined in-vitro conditions may present a viable opportunity to replace dying neurons. We adopted a previously published approach to differentiate Mesenchymal Stem Cells (MSCs) into DN using a 12-day protocol involving FGF-2, bFGF, SHH ligand and BDNF. While MSC-derived DNs have been characterized for neuronal markers and electrophysiological properties, we investigated store-operated calcium entry (SOCE) mechanisms of these DNs under normal conditions, and upon exposure to environmental neurotoxin, 1-methyl, 4-phenyl pyridinium ion (MPP+). Overall, we show that MSC-derived DNs are functional with regard to SOCE mechanisms, and MPP+ exposure dysregulates calcium signaling, making them vulnerable to neurodegeneration. Since in-vitro differentiation of MSCs into DNs is an important vehicle for PD disease modeling and regenerative medicine, the results of this study may help with understanding of the pathological mechanisms underlying PD.

Optimized staining and proliferation modeling methods for cell division monitoring using cell tracking dyes.

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.(1-5) Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of: stem and progenitor cell quiescence, proliferation and/or differentiation(6-8) antigen-driven membrane transfer(9) and/or precursor cell proliferation(3,4,10-18) and immune regulatory and effector cell function(1,18-21). Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes(1,2,11). "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins(4,16,18). Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class(2-4,16,18,24). The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are: Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies. Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 10(2) - 10(3) times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used. Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile. Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Image cytometry-based detection of aneuploidy by fluorescence in situ hybridization in suspension.

Cytogenetic abnormalities are important diagnostic and prognostic criteria for hematologic malignancies. Karyotyping and fluorescence in situ hybridization (FISH) are the conventional methods by which these abnormalities are detected. The sensitivity of these microscopy-based methods is limited by the abundance of the abnormal cells in the samples and therefore these analyses are commonly not applicable to minimal residual disease (MRD) stages. A flow cytometry-based imaging approach was developed to detect chromosomal abnormalities following FISH in suspension (FISH-IS), which enables the automated analysis of several log-magnitude higher number of cells compared with the microscopy-based approaches. This study demonstrates the applicability of FISH-IS for detecting numerical chromosome aberrations, establishes accuracy, and sensitivity of detection compared with conventional FISH, and feasibility to study procured clinical samples of acute myeloid leukemia (AML). Male and female healthy donor peripheral blood mononuclear cells hybridized with combinations of chromosome enumeration probes (CEP) 8, X, and Y served as models for disomy, monosomy, and trisomy. The sensitivity of detection of monosomies and trisomies amongst 20,000 analyzed cells was determined to be 1% with a high level of precision. A high correlation (R(2) = 0.99) with conventional FISH analysis was found based on the parallel analysis of diagnostic samples procured from 10 AML patients with trisomy 8 (+8). Additionally, FISH-IS analysis of samples procured at the time of clinical remission demonstrated the presence of residual +8 cells indicating that this approach may be used to detect MRD and associated chromosomal defects.