nAChRs Mediate Human Embryonic Stem Cell-Derived Endothelial Cells: Proliferation, Apoptosis, and Angiogenesis

Many patients with ischemic heart disease have cardiovascular risk factors such as cigarette smoking. We tested the effect of nicotine (a key component of cigarette smoking) on the therapeutic effects of human embryonic stem cell-derived endothelial cells (hESC-ECs).To induce endothelial cell differentiation, undifferentiated hESCs (H9 line) underwent 4-day floating EB formation and 8-day outgrowth differentiation in EGM-2 media. After 12 days, CD31(+) cells (13.7+/-2.5%) were sorted by FACScan and maintained in EGM-2 media for further differentiation. After isolation, these hESC-ECs expressed endothelial specific markers such as vWF (96.3+/-1.4%), CD31 (97.2+/-2.5%), and VE-cadherin (93.7+/-2.8%), form vascular-like channels, and incorporated DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL). Afterward, 5x10(6) hESC-ECs treated for 24 hours with nicotine (10(-8) M) or PBS (as control) were injected into the hearts of mice undergoing LAD ligation followed by administration for two weeks of vehicle or nicotine (100 microg/ml) in the drinking water. Surprisingly, bioluminescence imaging (BLI) showed significant improvement in the survival of transplanted hESC-ECs in the nicotine treated group at 6 weeks. Postmortem analysis confirmed increased presence of small capillaries in the infarcted zones. Finally, in vitro mechanistic analysis suggests activation of the MAPK and Akt pathways following activation of nicotinic acetylcholine receptors (nAChRs).This study shows for the first time that short-term systemic administrations of low dose nicotine can improve the survival of transplanted hESC-ECs, and enhance their angiogenic effects in vivo. Furthermore, activation of nAChRs has anti-apoptotic, angiogenic, and proliferative effects through MAPK and Akt signaling pathways

Functional and Transcriptional Characterization of Human Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Myocardial Infarction

BACKGROUND: Differentiation of human embryonic stem cells into endothelial cells (hESC-ECs) has the potential to provide an unlimited source of cells for novel transplantation therapies of ischemic diseases by supporting angiogenesis and vasculogenesis. However, the endothelial differentiation efficiency of the conventional embryoid body (EB) method is low while the 2-dimensional method of co-culturing with mouse embryonic fibroblasts (MEFs) require animal product, both of which can limit the future clinical application of hESC-ECs. Moreover, to fully understand the beneficial effects of stem cell therapy, investigators must be able to track the functional biology and physiology of transplanted cells in living subjects over time. METHODOLOGY: In this study, we developed an extracellular matrix (ECM) culture system for increasing endothelial differentiation and free from contaminating animal cells. We investigated the transcriptional changes that occur during endothelial differentiation of hESCs using whole genome microarray, and compared to human umbilical vein endothelial cells (HUVECs). We also showed functional vascular formation by hESC-ECs in a mouse dorsal window model. Moreover, our study is the first so far to transplant hESC-ECs in a myocardial infarction model and monitor cell fate using molecular imaging methods. CONCLUSION: Taken together, we report a more efficient method for derivation of hESC-ECs that express appropriate patterns of endothelial genes, form functional vessels in vivo, and improve cardiac function. These studies suggest that hESC-ECs may provide a novel therapy for ischemic heart disease in the future

Distinct Roles of MicroRNA-1 and -499 in Ventricular Specification and Functional Maturation of Human Embryonic Stem Cell-Derived Cardiomyocytes

BACKGROUND: MicroRNAs (miRs) negatively regulate transcription and are important determinants of normal heart development and heart failure pathogenesis. Despite the significant knowledge gained in mouse studies, their functional roles in human (h) heart remain elusive. METHODS AND RESULTS: We hypothesized that miRs that figure prominently in cardiac differentiation are differentially expressed in differentiating, developing, and terminally mature human cardiomyocytes (CMs). As a first step, we mapped the miR profiles of human (h) embryonic stem cells (ESCs), hESC-derived (hE), fetal (hF) and adult (hA) ventricular (V) CMs. 63 miRs were differentially expressed between hESCs and hE-VCMs. Of these, 29, including the miR-302 and -371/372/373 clusters, were associated with pluripotency and uniquely expressed in hESCs. Of the remaining miRs differentially expressed in hE-VCMs, 23 continued to express highly in hF- and hA-VCMs, with miR-1, -133, and -499 displaying the largest fold differences; others such as miR-let-7a, -let-7b, -26b, -125a and -143 were non-cardiac specific. Functionally, LV-miR-499 transduction of hESC-derived cardiovascular progenitors significantly increased the yield of hE-VCMs (to 72% from 48% of control; p0.05). By contrast, LV-miR-1 transduction did not bias the yield (p>0.05) but decreased APD and hyperpolarized RMP/MDP in hE-VCMs due to increased I(to), I(Ks) and I(Kr), and decreased I(f) (p<0.05) as signs of functional maturation. Also, LV-miR-1 but not -499 augmented the immature Ca(2+) transient amplitude and kinetics. Molecular pathway analyses were performed for further insights. CONCLUSION: We conclude that miR-1 and -499 play differential roles in cardiac differentiation of hESCs in a context-dependent fashion. While miR-499 promotes ventricular specification of hESCs, miR-1 serves to facilitate electrophysiological maturation.published_or_final_versio

Women's colposcopy experience and preferences: a mixed methods study

<p>Abstract</p> <p>Background</p> <p>The colposcopy service is a key component in the UK Cervical Screening Programme. Over 120,000 women are referred to the service annually, however up to 25% of women fail to attend their appointment. Little is known about patients' preferences for colposcopic investigation and treatment. This study aims to investigate women's experience of colposcopy, to identify patients' preferences for aspects of appointments within the colposcopy service, and to make suggestions for service improvement.</p> <p>Methods/Design</p> <p>This study has been designed as a two stage, mixed method project. Stage one will involve in-depth interviews with new colposcopy patients to ascertain their experience of colposcopy services. This qualitative stage will generate factors thought to be important by service users in their experience. Stage two will utilise a choice based quantitative technique to identify women's preferences and determine the representativeness of factors generated through the interviews.</p> <p>The initial stage of in-depth interviews will be conducted with patients who are newly referred to colposcopy clinics to investigate the experience that they have of the referral process and appointment attendance. The outcome of these interviews will be analysed qualitatively using Framework analysis. Factors found to be important in women's experience will be extracted and used to construct a choice based questionnaire.</p> <p>The discrete choice experiment (questionnaire) will apply a best-worst technique through scenario-based questions to find women's relative preferences for different aspects of the service. It will be offered to women attending follow-up appointments at two colposcopy clinics in the West Midlands. Women will complete the questionnaire whilst they wait for their appointment, or, if they prefer, will take it home to complete in private. Women who do not attend their appointment will be posted the research information and questionnaire. The questionnaire analysis will use a weighted least squares regression technique for each best/worst pair. The accept/reject 'would you attend this appointment' question will be analysed using a random effects logit model.</p> <p>Discussion</p> <p>Colposcopy is a common procedure and one that is associated with raised anxiety among women experiencing the service. Little is known about women's experience of the service or their preferences for service delivery. The outcomes of the study will comprise a description of women's experience of colposcopy and establishing their preferences for how aspects of the service should be provided. Women's preferences will be fed back to service providers to enable improvements to the service to be made.</p

Human germ cell differentiation from fetal- and adult-derived induced pluripotent stem cells

Historically, our understanding of molecular genetic aspects of human germ cell development has been limited, at least in part due to inaccessibility of early stages of human development to experimentation. However, the derivation of pluripotent stem cells may provide the necessary human genetic system to study germ cell development. In this study, we compared the potential of human induced pluripotent stem cells (iPSCs), derived from adult and fetal somatic cells to form primordial and meiotic germ cells, relative to human embryonic stem cells. We found that ∼5% of human iPSCs differentiated to primordial germ cells (PGCs) following induction with bone morphogenetic proteins. Furthermore, we observed that PGCs expressed green fluorescent protein from a germ cell-specific reporter and were enriched for the expression of endogenous germ cell-specific proteins and mRNAs. In response to the overexpression of intrinsic regulators, we also observed that iPSCs formed meiotic cells with extensive synaptonemal complexes and post-meiotic haploid cells with a similar pattern of ACROSIN staining as observed in human spermatids. These results indicate that human iPSCs derived from reprogramming of adult somatic cells can form germline cells. This system may provide a useful model for molecular genetic studies of human germline formation and pathology and a novel platform for clinical studies and potential therapeutical applications

The Manchester International Consensus Group recommendations for the management of gynecological cancers in Lynch syndrome.

PURPOSE: There are no internationally agreed upon clinical guidelines as to which women with gynecological cancer would benefit from Lynch syndrome screening or how best to manage the risk of gynecological cancer in women with Lynch syndrome. The Manchester International Consensus Group was convened in April 2017 to address this unmet need. The aim of the Group was to develop clear and comprehensive clinical guidance regarding the management of the gynecological sequelae of Lynch syndrome based on existing evidence and expert opinion from medical professionals and patients. METHODS: Stakeholders from Europe and North America worked together over a two-day workshop to achieve consensus on best practice. RESULTS: Guidance was developed in four key areas: (1) whether women with gynecological cancer should be screened for Lynch syndrome and (2) how this should be done, (3) whether there was a role for gynecological surveillance in women at risk of Lynch syndrome, and (4) what preventive measures should be recommended for women with Lynch syndrome to reduce their risk of gynecological cancer. CONCLUSION: This document provides comprehensive clinical guidance that can be referenced by both patients and clinicians so that women with Lynch syndrome can expect and receive appropriate standards of care

Burning in Banksia Woodlands: How Does the Fire-Free Period Influence Reptile Communities?

Fire is an important management tool for both hazard reduction burning and maintenance of biodiversity. The impact of time since last fire on fauna is an important factor to understand as land managers often aim for prescribed burning regimes with specific fire-free intervals. However, our current understanding of the impact of time since last fire on fauna is largely unknown and likely dependent on vegetation type. We examined the responses of reptiles to fire age in banksia woodlands, and the interspersed melaleuca damplands among them, north of Perth, Western Australia, where the current prescribed burning regime is targeting a fire-free period of 8–12 years. The response of reptiles to fire was dependent on vegetation type. Reptiles were generally more abundant (e.g. Lerista elegans and Ctenophorus adelaidensis) and specious in banksia sites. Several species (e.g. Menetia greyii, Cryptoblepharus buchananii) preferred long unburnt melaleuca sites (>16 years since last fire, YSLF) compared to recently burnt sites (<12 YSLF). Several of the small elapids (e.g. the WA priority listed species Neelaps calonotus) were only detected in older-aged banksia sites (>16 YSLF). The terrestrial dragon C. adelaidensis and the skink Morethia obscura displayed a strong response to fire in banksia woodlands only. Highest abundances of the dragon were detected in the recently burnt (<7 YSLF) and long unburnt (>35 YSLF) banksia woodlands, while the skink was more abundant in older sites. Habitats from a range of fire ages are required to support the reptiles we detected, especially the longer unburnt (>16 YSLF) melaleuca habitat. Current burning prescriptions are reducing the availability of these older habitats

Transcriptome Alteration in the Diabetic Heart by Rosiglitazone: Implications for Cardiovascular Mortality

BACKGROUND: Recently, the type 2 diabetes medication, rosiglitazone, has come under scrutiny for possibly increasing the risk of cardiac disease and death. To investigate the effects of rosiglitazone on the diabetic heart, we performed cardiac transcriptional profiling and imaging studies of a murine model of type 2 diabetes, the C57BL/KLS-lepr(db)/lepr(db) (db/db) mouse. METHODS AND FINDINGS: We compared cardiac gene expression profiles from three groups: untreated db/db mice, db/db mice after rosiglitazone treatment, and non-diabetic db/+ mice. Prior to sacrifice, we also performed cardiac magnetic resonance (CMR) and echocardiography. As expected, overall the db/db gene expression signature was markedly different from control, but to our surprise was not significantly reversed with rosiglitazone. In particular, we have uncovered a number of rosiglitazone modulated genes and pathways that may play a role in the pathophysiology of the increase in cardiac mortality as seen in several recent meta-analyses. Specifically, the cumulative upregulation of (1) a matrix metalloproteinase gene that has previously been implicated in plaque rupture, (2) potassium channel genes involved in membrane potential maintenance and action potential generation, and (3) sphingolipid and ceramide metabolism-related genes, together give cause for concern over rosiglitazone's safety. Lastly, in vivo imaging studies revealed minimal differences between rosiglitazone-treated and untreated db/db mouse hearts, indicating that rosiglitazone's effects on gene expression in the heart do not immediately turn into detectable gross functional changes. CONCLUSIONS: This study maps the genomic expression patterns in the hearts of the db/db murine model of diabetes and illustrates the impact of rosiglitazone on these patterns. The db/db gene expression signature was markedly different from control, and was not reversed with rosiglitazone. A smaller number of unique and interesting changes in gene expression were noted with rosiglitazone treatment. Further study of these genes and molecular pathways will provide important insights into the cardiac decompensation associated with both diabetes and rosiglitazone treatment

An experimental study exploring the impact of vignette gender on the quality of university students’ mental health first aid for peers with symptoms of depression

Background University students have high rates of depression, and friends are often the most commonly-used source of support for emotional distress in this population. This study aimed to explore students’ ability to provide effective support for their peers with depressive symptoms and the factors influencing the quality of their mental health first aid (MHFA) skills, including students’ gender, course of study, and gender of student experiencing depression. Methods Via an online survey, students at two British universities (N = 483) were quasi-randomly allocated to view a video vignette of either a male or female student depicting symptoms of depression. An open-ended question probed MHFA actions they would take to help the vignette character, which were rated using a standardised scoring scheme based on MHFA guidelines. Results Students reported low MHFA scores (mean 2.89, out of possible 12). The most commonly reported action was provision of support and information, but only eight (1.6 %) students stated an intention to assess risk of harm. Those studying clinically non-relevant degrees with limited mental health content reported poorer MHFA (p = <0.001) and were less confident about their ability to support a friend with depression (p = 0.04). There was no main effect of vignette gender, but within the group of students on non-relevant courses the male vignette received significantly poorer MHFA than the female vignette (p = 0.02). A significant three-way interaction found that male participants studying non-relevant degrees who viewed a male vignette had poorer MHFA compared to females studying non-relevant degrees who viewed the female vignette (p = 0.005). Conclusions Most students lack the necessary MHFA skills to support friends suffering from symptoms of depression, or to help them get appropriate support and prevent risk of harm. Students on courses which do not include mental health related content are particularly ill-equipped to support male students, with male students receiving the poorest quality MHFA from fellow male students on these courses. MHFA training has the potential to improve outcomes for students with depression, and could have a valuable role in reducing the excess risk of harm seen in male students

The Role of DNA Barcodes in Understanding and Conservation of Mammal Diversity in Southeast Asia

Southeast Asia is recognized as a region of very high biodiversity, much of which is currently at risk due to habitat loss and other threats. However, many aspects of this diversity, even for relatively well-known groups such as mammals, are poorly known, limiting ability to develop conservation plans. This study examines the value of DNA barcodes, sequences of the mitochondrial COI gene, to enhance understanding of mammalian diversity in the region and hence to aid conservation planning.DNA barcodes were obtained from nearly 1900 specimens representing 165 recognized species of bats. All morphologically or acoustically distinct species, based on classical taxonomy, could be discriminated with DNA barcodes except four closely allied species pairs. Many currently recognized species contained multiple barcode lineages, often with deep divergence suggesting unrecognized species. In addition, most widespread species showed substantial genetic differentiation across their distributions. Our results suggest that mammal species richness within the region may be underestimated by at least 50%, and there are higher levels of endemism and greater intra-specific population structure than previously recognized.DNA barcodes can aid conservation and research by assisting field workers in identifying species, by helping taxonomists determine species groups needing more detailed analysis, and by facilitating the recognition of the appropriate units and scales for conservation planning