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2.  Coronary Artery Perforation following PCI: An Interesting Finding into the Pericardial Space 
Coronary artery perforation is a known complication of percutaneous coronary intervention and potentially life threatening. Normally, these perforations are small and localized. We report the successful surgical management of a coronary artery perforation following stent insertion with extrusion of an 8-cm endarterectomy length of the circumflex coronary artery with a brief review of the recent literature.
doi:10.1055/s-0033-1343359
PMCID: PMC3830569  PMID: 24436619
coronary artery perforation; percutaneous coronary intervention; endarterectomy; coronary artery; cardiac surgery
3.  The immune cell infiltrate populating meningiomas is composed of mature, antigen-experienced T and B cells 
Neuro-Oncology  2013;15(11):1479-1490.
Background
Meningiomas often harbor an immune cell infiltrate that can include substantial numbers of T and B cells. However, their phenotype and characteristics remain undefined. To gain a deeper understanding of the T and B cell repertoire in this tumor, we characterized the immune infiltrate of 28 resected meningiomas representing all grades.
Methods
Immunohistochemistry was used to grossly characterize and enumerate infiltrating lymphocytes. A molecular analysis of the immunoglobulin variable region of tumor-infiltrating B cells was used to characterize their antigen experience. Flow cytometry of fresh tissue homogenate and paired peripheral blood lymphocytes was used to identify T cell phenotypes and characterize the T cell repertoire.
Results
A conspicuous B and T cell infiltrate, primarily clustered in perivascular spaces, was present in the microenvironment of most tumors examined. Characterization of 294 tumor-infiltrating B cells revealed clear evidence of antigen experience, in that the cardinal features of an antigen-driven B cell response were present. Meningiomas harbored populations of antigen-experienced CD4+ and CD8+ memory/effector T cells, regulatory T cells, and T cells expressing the immune checkpoint molecules PD-1 and Tim-3, indicative of exhaustion. All of these phenotypes were considerably enriched relative to their frequency in the circulation. The T cell repertoire in the tumor microenvironment included populations that were not reflected in paired peripheral blood.
Conclusion
The tumor microenvironment of meningiomas often includes postgerminal center B cell populations. These tumors invariably include a selected, antigen-experienced, effector T cell population enriched by those that express markers of an exhausted phenotype.
doi:10.1093/neuonc/not110
PMCID: PMC3813416  PMID: 23978377
B cell; meningioma; T cell; tumor-infiltrating lymphocytes
4.  A Validated Age-Related Normative Model for Male Total Testosterone Shows Increasing Variance but No Decline after Age 40 Years 
PLoS ONE  2014;9(10):e109346.
The diagnosis of hypogonadism in human males includes identification of low serum testosterone levels, and hence there is an underlying assumption that normal ranges of testosterone for the healthy population are known for all ages. However, to our knowledge, no such reference model exists in the literature, and hence the availability of an applicable biochemical reference range would be helpful for the clinical assessment of hypogonadal men. In this study, using model selection and validation analysis of data identified and extracted from thirteen studies, we derive and validate a normative model of total testosterone across the lifespan in healthy men. We show that total testosterone peaks [mean (2.5–97.5 percentile)] at 15.4 (7.2–31.1) nmol/L at an average age of 19 years, and falls in the average case [mean (2.5–97.5 percentile)] to 13.0 (6.6–25.3) nmol/L by age 40 years, but we find no evidence for a further fall in mean total testosterone with increasing age through to old age. However we do show that there is an increased variation in total testosterone levels with advancing age after age 40 years. This model provides the age related reference ranges needed to support research and clinical decision making in males who have symptoms that may be due to hypogonadism.
doi:10.1371/journal.pone.0109346
PMCID: PMC4190174  PMID: 25295520
5.  Fertility preservation for girls and young women with cancer: population-based validation of criteria for ovarian tissue cryopreservation 
The Lancet. Oncology  2014;15(10):1129-1136.
Summary
Background
Ovarian tissue cryopreservation with later reimplantation has been shown to preserve fertility in adult women, but this approach remains unproven and experimental in children and adolescents. We aimed to assess the use of the Edinburgh selection criteria for ovarian tissue cryopreservation in girls and young women with cancer to determine whether we are offering this invasive procedure to the patients who are most at risk of premature ovarian insufficiency.
Methods
Cryopreservation of ovarian tissue has been selectively offered to girls and young women with cancer who met the Edinburgh selection criteria since 1996. Between Jan 1, 1996, and June 30, 2012, 410 female patients younger than 18 years at diagnosis were treated for cancer (including leukaemia and brain tumours) at the Edinburgh Children's Cancer Centre, which serves the whole South East of Scotland region. We determined the ovarian status of these patients from review of clinical records and classified them as having premature ovarian insufficiency or not, or as unable to be determined. Patients younger than 12 years at time of data cutoff (Jan 31, 2013) were excluded from the analysis.
Findings
34 (8%) of the 410 patients met the Edinburgh selection criteria and were offered ovarian tissue cryopreservation before starting cancer treatment. 13 patients declined the procedure and 21 consented, and the procedure was completed successfully in 20 patients. Of the 20 patients who had ovarian tissue successfully cryopreserved, 14 were available for assessment of ovarian function. Of the 13 patients who had declined the procedure, six were available for assessment of ovarian function. Median age at the time of follow-up for the 20 assessable patients was 16·9 years (IQR 15·5–21·8). Of the 14 assessable patients who had successfully undergone ovarian cryopreservation, six had developed premature ovarian insufficiency at a median age of 13·4 years (IQR 12·5–14·6), one of whom also had a natural pregnancy. Of the six assessable patients who had declined the procedure, one had developed premature ovarian insufficiency. Assessment of ovarian function was possible for 141 of the 376 patients who were not offered cryopreservation; one of these patients had developed premature ovarian insufficiency. The cumulative probability of developing premature ovarian insufficiency after treatment was completed was significantly higher for patients who met the criteria for ovarian tissue cryopreservation than for those who did not (15-year probability 35% [95% CI 10–53] vs 1% [0–2]; p<0·0001; hazard ratio 56·8 [95% CI 6·2–521·6] at 10 years).
Interpretation
The results of this analysis show that the Edinburgh selection criteria accurately identify the few girls and young women who will develop premature ovarian insufficiency, and validate their use for selection of patients for ovarian tissue cryopreservation. Further follow-up of this cohort of patients is likely to allow refinement of the criteria for this experimental procedure in girls and young women with cancer.
Funding
UK Medical Research Council.
doi:10.1016/S1470-2045(14)70334-1
PMCID: PMC4153375  PMID: 25130994
6.  A highly-sensitive anti-Müllerian hormone assay improves analysis of ovarian function following chemotherapy for early breast cancer☆ 
European Journal of Cancer  2014;50(14):2367-2374.
Aim
Anti-Müllerian hormone (AMH) shows promise as a biomarker of the ovarian reserve but current assays are insufficiently sensitive to allow assessment of this post-chemotherapy in most women. We have assessed a new highly sensitive AMH assay (Ansh picoAMH) in the evaluation of ovarian activity in women with very low ovarian reserve after chemotherapy.
Methods
A prospective cohort and an independent validation cohort of premenopausal women with early breast cancer (eBC) were recruited at the time of diagnosis (combined n = 98), and ovarian reserve markers 2–5 years later following chemotherapy were assessed in relation to menstrual activity.
Results
The picoAMH assay had a limit of detection of 7.5 pg/ml. AMH clearly distinguished women with ongoing menses from those with amenorrhoea at 2 years after diagnosis (mean 522 ± 169 versus 8.9 ± 1.3 pg/ml, P < 0.0001) with high predictive value for continuing menses or amenorrhoea for the subsequent 3 years. AMH was detectable in more women than using a previous assay (P = 0.004). Other markers of the ovarian reserve (follicle-stimulating hormone (FSH), inhibin B) were also of discriminatory value but to lesser extents. This finding was validated in a second, independent cohort of women treated for eBC.
Conclusion
The 10-fold increased assay sensitivity showed very clear distinction between groups based on ovarian activity with excellent prediction of future menses or amenorrhoea. This will improve assessment of post-chemotherapy ovarian function in women and may aid treatment decisions.
doi:10.1016/j.ejca.2014.06.011
PMCID: PMC4166459  PMID: 25027307
AMH; Breast cancer; Ovarian function; Chemotherapy; Ovarian reserve
7.  Ovarian germline stem cells 
It has long been established that germline stem cells (GSCs) are responsible for lifelong gametogenesis in males, and some female invertebrates (for example, Drosophila) and lower vertebrates (for example, teleost fish and some prosimians) also appear to rely on GSCs to replenish their oocyte reserve in adulthood. However, the presence of such cells in the majority of female mammals is controversial, and the idea of a fixed ovarian reserve determined at birth is the prevailing belief among reproductive biologists. However, accumulating evidence demonstrates the isolation and culture of putative GSCs from the ovaries of adult mice and humans. Live offspring have been reportedly produced from the culture of adult mouse GSCs, and human GSCs formed primordial follicles using a mouse xenograft model. If GSCs were present in adult female ovaries, it could be postulated that the occurrence of menopause is not due to the exhaustion of a fixed supply of oocytes but instead is a result of GSC and somatic cell aging. Alternatively, they may be benign under normal physiological conditions. If their existence were confirmed, female GSCs could have many potential applications in both basic science and clinical therapies. GSCs not only may provide a valuable model for germ cell development and maturation but may have a role in the field of fertility preservation, with women potentially being able to store GSCs or GSC-derived oocytes from their own ovaries prior to infertility-inducing treatments. Essential future work in this field will include further independent corroboration of the existence of GSCs in female mammals and the demonstration of the production of mature competent oocytes from GSCs cultured entirely in vitro.
doi:10.1186/scrt487
PMCID: PMC4282152  PMID: 25157949
8.  Docetaxel induces moderate ovarian toxicity in mice, primarily affecting granulosa cells of early growing follicles 
Molecular Human Reproduction  2014;20(10):948-959.
Advances in cancer therapy have focused attention on the quality of life of cancer survivors. Since infertility is a major concern following chemotherapy, it is important to characterize the drug-specific damage to the reproductive system to help find appropriate protective strategies. This study investigates the damage on neonatal mouse ovary maintained in vitro for 6 days, and exposed for 24 h (on Day 2) to clinically relevant doses of Docetaxel (DOC; low: 0.1 µM, mid: 1 µM, high: 10 µM). Furthermore, the study explores the putative protective action exerted by Tri-iodothyronine (T3; 10−7 M). At the end of culture, morphological analyses and follicle counts showed that DOC negatively impacts on early growing follicles, decreasing primary follicle number and severely affecting health at the transitional and primary stages. Poor follicle health was mainly due to effects on granulosa cells, indicating that the effects of DOC on oocytes were likely to be secondary to granulosa cell damage. DOC damages growing follicles specifically, with no direct effect on the primordial follicle reserve. Immunostaining and western blotting showed that DOC induces activation of intrinsic, type II apoptosis in ovarian somatic cells; increasing the levels of cleaved caspase 3, cleaved caspase 8, Bax and cleaved poly(ADP-ribose) polymerase, while also inducing movement of cytochrome C from mitochondria into the cytosol. T3 did not prevent the damage induced by the low dose of DOC. These results demonstrated that DOC induces a gonadotoxic effect on the mouse ovary through induction of somatic cell apoptosis, with no evidence of direct effects on the oocyte, and that the damaging effect is not mitigated by T3.
doi:10.1093/molehr/gau057
PMCID: PMC4172173  PMID: 25080441
apoptosis; docetaxel; mouse; ovarian follicles; thyroid hormone
9.  Interaction between access choice and pharmacotherapy for coronary intervention: the results of a UK survey 
Open Heart  2014;1(1):e000094.
Introduction
Percutaneous coronary intervention (PCI) has changed significantly over the past decade with the uptake of radial access and the development of newer and more potent antiplatelets and safer antithrombins. This survey examined the default access route and pharmacology choice and their interaction in UK interventional practice.
Methods
An email-based survey invited interventional cardiologists to answer questions regarding arterial access and pharmacology use during PCI. Respondents were categorised into femoral, radial and radial+ (if the other radial was used rather than femoral if the right radial attempt failed). Data were analysed using χ2 or the Student t test.
Results
81% of the 204 respondents reported the radial artery as their default access site with a significant interaction between years since qualification and access choice (21.1 years for radial+ vs 23 years for radial (p=0.027) vs 26.6 years for femoral (p=0.013) vs radial (p=0.0005) vs radial+). There were 19 different combinations of access and pharmacology reported. For non-ST elevation myocardial infarction PCI, there was a significant trend for radial+ and radial operators to favour ticagrelor or tailored therapy versus femoral operators (54.8% vs 47.8% vs 35%, respectively, p=0.018). For primary PCI (PPCI), radial+ and radial operators were much more likely to choose ticagrelor or prasugrel than femoral operators (77.2% (p<0.001) vs 73.9% (p=0.023) vs 50%, respectively (p<0.0001) for trend). For PPCI, glycoprotein inhibitor use was similar between groups (26.1% vs 25%, not significant); radial operators were much more likely to choose bivalirudin (52.8% vs 10%, p<0.0001) and much less likely to use heparin only (19.8% vs 65%, p<0.0001) than femoral operators.
Conclusions
There is a significant interaction between years since qualification and access choice. Although there is no established consensus on access site or drugs, default radial operators are significantly more likely to utilise new generation antiplatelets and bivalirudin than femoral operators.
doi:10.1136/openhrt-2014-000094
PMCID: PMC4195931  PMID: 25332809
10.  Phosphatidylinositol-4,5-bisphosphate: Targeted Production and Signaling 
Summary
Phosphatidylinositol-4,5-bisphosphate (PI4,5P2) is a key lipid signaling molecule that regulates a vast array of biological activities. PI4,5P2 can directly act as a messenger or can be utilized as a precursor to generate other messengers: inositol trisphosphate (IP3), diacylglycerol (DAG) or phosphatidylinositol-3,4,5-trisphosphate (PI3,4,5P3). PI4,5P2 interacts with hundreds of different effector proteins. The enormous diversity of PI4,5P2 effector proteins and the spatio-temporal control of PI4,5P2 generation allows PI4,5P2 signaling to control a broad spectrum of cellular functions. PI4,5P2 is synthesized by phosphatidylinositol phosphate kinases (PIPKs). The array of PIPKs in cells enables their targeting to specific sub-cellular compartments through interactions with targeting factors that are often PI4,5P2 effectors. These interactions are a mechanism to define spatial and temporal PI4,5P2 synthesis and the specificity of PI4,5P2 signaling. In turn the regulation of PI4,5P2 effectors at specific cellular compartments has implications for understanding how PI4,5P2 controls cellular processes and its role in diseases.
doi:10.1002/bies.201200171
PMCID: PMC3882169  PMID: 23575577
Phosphatidylinositol-4,5-bisphosphate; phosphatidylinositol phosphate kinase; PI4,5P2 effector; lipid messenger
11.  Inhibition of phosphatase and tensin homologue (PTEN) in human ovary in vitro results in increased activation of primordial follicles but compromises development of growing follicles 
Molecular Human Reproduction  2014;20(8):736-744.
In the mammalian ovary a small number of follicles are steadily recruited from the quiescent pool to undergo development. Follicle loss, maintenance and growth are strictly controlled by complex molecular interactions including the phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt) signalling pathway. Stimulation of PI3K promotes phosphorylation of Akt resulting in follicle survival and activation of growth whereas this pathway is suppressed by the actions of the phosphatase and tensin homologue (PTEN). The aim of this study was to determine the effect of dipotassium bisperoxo(5-hydroxypyridine-2-carboxyl)oxovanadate (bpV), a reversible inhibitor of PTEN, on the activation, survival and development of human ovarian follicles in vitro. Biopsied ovarian tissue fragments were obtained from 17 women aged 23–46 years and exposed to 1 µM bpV(HOpic) (n = 146) or control medium (n = 128) for 24 h. Media were then replaced with control medium and all tissue incubated for a further 5 days. Ovarian tissue from each treatment group was fixed after the initial 24 h culture period and phosphorylated Akt was quantified by western blotting. After 6 days incubation all tissue fragments were inspected under light microscopy and any secondary follicles ≥100 µm isolated. Isolated follicles were cultured individually in control medium supplemented with 100 ng/ml recombinant human activin A. Tissue fragments without follicles suitable for isolation were fixed and processed for histological and immunohistochemical analysis. During 6 days culture, follicle activation occurred in tissue samples from both treatment groups but with significantly more follicles progressing to the secondary stage of development in the presence of 1 µM bpV(HOpic) compared with control (31 versus 16%; P < 0.05). Increased activation was associated with increased Akt phosphorylation and increased nuclear export of FOXO3. However isolated and cultured follicles that had been exposed to bpV(HOpic) showed limited growth and reduced survival compared with follicles from control fragments (P < 0.05). This study demonstrates that inhibition of PTEN with bpV(HOpic) affects human ovarian follicle development by promoting the initiation of follicle growth and development to the secondary stage, as in rodent species, but severely compromises the survival of isolated secondary follicles.
doi:10.1093/molehr/gau037
PMCID: PMC4106636  PMID: 24830779
PTEN; in vitro; human; follicle; ovary
12.  Characterization of the Metabolic and Physiologic Response from Chromium Supplementation in Subjects with Type 2 Diabetes 
OBJECTIVE
To provide a comprehensive evaluation of chromium (Cr) supplementation on metabolic parameters in a cohort of Type 2 DM subjects representing a wide phenotype range and to evaluate changes in “responders” and “non-responders”.
DESIGN
After pre-intervention testing to assess glycemia, insulin sensitivity (assessed by euglycemic clamps), Cr status, body composition, subjects were randomized in a double-blind fashion to placebo or 1,000 μg Cr. A sub-study was performed to evaluate 24 hour energy balance/substrate oxidation and myocellular/intra-hepatic lipid content.
RESULTS
There was not a consistent effect of chromium supplementation to improve insulin action across all phenotypes. Insulin sensitivity was negatively correlated to soleus and tibialis muscle intramyocellular lipids and intra-hepatic lipid content. Myocellular lipids were significantly lower in subjects randomized to Cr. At pre-intervention, “responders”, defined as insulin sensitivity change from baseline > 10%, had significantly lower insulin sensitivity and higher fasting glucose and A1c when compared to placebo and “non-responders”, i.e. insulin sensitivity change from baseline < 10%. Clinical response was significantly correlated (p < 0.001) to the baseline insulin sensitivity, fasting glucose and A1c. There was no difference in Cr status between “responders”, and “non-responders”.
CONCLUSIONS
Clinical response to chromium is more likely in insulin resistant subjects who have more elevated fasting glucose and A1c levels. Cr may reduce myocellular lipids and enhance insulin sensitivity in subjects with type 2 DM independent of effects on weight or hepatic glucose production. Thus, modulation of lipid metabolism by Cr in peripheral tissues may represent a novel mechanism of action.
doi:10.1016/j.metabol.2009.09.023
PMCID: PMC4020630  PMID: 20022616
Chromium; nutrition; glucose; trace minerals
13.  Endosomal Type Iγ PIP 5-Kinase Controls EGF Receptor Lysosomal Sorting 
Developmental cell  2013;25(2):144-155.
Summary
Endosomal trafficking and degradation of epidermal growth factor receptor (EGFR) play an essential role in control of its signaling. Phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P2) is an established regulator of endocytosis, whereas PtdIns3P modulates endosomal trafficking. However, here we demonstrate that type Igamma phosphatidylinositol phosphate 5-kinase i5 (PIPKIγi5), an enzyme that synthesizes PtdIns4,5P2, controls endosome to lysosome sorting of EGFR. In this pathway, PIPKIγi5 interacts with sorting nexin 5 (SNX5), a protein that binds PtdIns4,5P2 and other phosphoinositides. PIPKIγi5 and SNX5 localize to endosomes, and loss of either protein blocks EGFR sorting into intraluminal vesicles (ILVs) of the multivesicular body (MVB). Loss of ILV sorting greatly enhances and prolongs EGFR signaling. PIPKIγi5 and SNX5 prevent Hrs ubiquitination and this facilitates the Hrs association with EGFR that is required for ILV sorting. These findings reveal that PIPKIγi5 and SNX5 form a unique signaling nexus that controls EGFR endosomal sorting, degradation, and signaling.
doi:10.1016/j.devcel.2013.03.010
PMCID: PMC3740164  PMID: 23602387
14.  Antenatal architecture and activity of the human heart 
Interface Focus  2013;3(2):20120065.
We construct the components for a family of computational models of the electrophysiology of the human foetal heart from 60 days gestational age (DGA) to full term. This requires both cell excitation models that reconstruct the myocyte action potentials, and datasets of cardiac geometry and architecture. Fast low-angle shot and diffusion tensor magnetic resonance imaging (DT-MRI) of foetal hearts provides cardiac geometry with voxel resolution of approximately 100 µm. DT-MRI measures the relative diffusion of protons and provides a measure of the average intravoxel myocyte orientation, and the orientation of any higher order orthotropic organization of the tissue. Such orthotropic organization in the adult mammalian heart has been identified with myocardial sheets and cleavage planes between them. During gestation, the architecture of the human ventricular wall changes from being irregular and isotropic at 100 DGA to an anisotropic and orthotropic architecture by 140 DGA, when it has the smooth, approximately 120° transmural change in myocyte orientation that is characteristic of the adult mammalian ventricle. The DT obtained from DT-MRI provides the conductivity tensor that determines the spread of potential within computational models of cardiac tissue electrophysiology. The foetal electrocardiogram (fECG) can be recorded from approximately 60 DGA, and RR, PR and QT intervals between the P, R, Q and T waves of the fECG can be extracted by averaging from approximately 90 DGA. The RR intervals provide a measure of the pacemaker rate, the QT intervals an index of ventricular action potential duration, and its rate-dependence, and so these intervals constrain and inform models of cell electrophysiology. The parameters of models of adult human sinostrial node and ventricular cells that are based on adult cell electrophysiology and tissue molecular mapping have been modified to construct preliminary models of foetal cell electrophysiology, which reproduce these intervals from fECG recordings. The PR and QR intervals provide an index of conduction times, and hence propagation velocities (approx. 1–10 cm s−1, increasing during gestation) and so inform models of tissue electrophysiology. Although the developing foetal heart is small and the cells are weakly coupled, it can support potentially lethal re-entrant arrhythmia.
doi:10.1098/rsfs.2012.0065
PMCID: PMC3638472  PMID: 24427520
foetal heart development; myocardial architecture; computational modelling; diffusion tensor magnetic resonance imaging; foetal ECG; cardiac electrophysiology
15.  Fetal Cyclophosphamide Exposure Induces Testicular Cancer and Reduced Spermatogenesis and Ovarian Follicle Numbers in Mice 
PLoS ONE  2014;9(4):e93311.
Exposure to radiation during fetal development induces testicular germ cell tumors (TGCT) and reduces spermatogenesis in mice. However, whether DNA damaging chemotherapeutic agents elicit these effects in mice remains unclear. Among such agents, cyclophosphamide (CP) is currently used to treat breast cancer in pregnant women, and the effects of fetal exposure to this drug manifested in the offspring must be better understood to offer such patients suitable counseling. The present study was designed to determine whether fetal exposure to CP induces testicular cancer and/or gonadal toxicity in 129 and in 129.MOLF congenic (L1) mice. Exposure to CP on embryonic days 10.5 and 11.5 dramatically increased TGCT incidence to 28% in offspring of 129 mice (control value, 2%) and to 80% in the male offspring of L1 (control value 33%). These increases are similar to those observed in both lines of mice by radiation. In utero exposure to CP also significantly reduced testis weights at 4 weeks of age to ∼70% of control and induced atrophic seminiferous tubules in ∼30% of the testes. When the in utero CP-exposed 129 mice reached adulthood, there were significant reductions in testicular and epididymal sperm counts to 62% and 70%, respectively, of controls. In female offspring, CP caused the loss of 77% of primordial follicles and increased follicle growth activation. The results indicate that i) DNA damage is a common mechanism leading to induction of testicular cancer, ii) increased induction of testis cancer by external agents is proportional to the spontaneous incidence due to inherent genetic susceptibility, and iii) children exposed to radiation or DNA damaging chemotherapeutic agents in utero may have increased risks of developing testis cancer and having reduced spermatogenic potential or diminished reproductive lifespan.
doi:10.1371/journal.pone.0093311
PMCID: PMC3972108  PMID: 24691397
16.  Identification of the Niche and Phenotype of the First Human Hematopoietic Stem Cells 
Stem Cell Reports  2014;2(4):449-456.
Summary
In various vertebrate species, the dorsal aorta (Ao) is the site of specification of adult hematopoietic stem cells (HSCs). It has been observed that the upregulation of essential hematopoietic transcription factors and the formation of specific intra-aortic hematopoietic cell clusters occur predominantly in the ventral domain of the Ao (AoV). In the mouse, the first HSCs emerge in the AoV. Here, we demonstrate that in the human embryo the first definitive HSCs also emerge asymmetrically and are localized to the AoV, which thus identifies a functional niche for developing human HSCs. Using magnetic cell separation and xenotransplantations, we show that the first human HSCs are CD34+VE-cadherin+CD45+C-KIT+THY-1+Endoglin+RUNX1+CD38−/loCD45RA−. This population harbors practically all committed hematopoietic progenitors and is underrepresented in the dorsal domain of the Ao (AoD) and urogenital ridges (UGRs). The present study provides a foundation for analysis of molecular mechanisms underpinning embryonic specification of human HSCs.
Highlights
•The first human HSCs develop in the AoV•These cells are CD34+VE-cadherin+CD45+C-KIT+THY-1+Endoglin+RUNX1+CD38−/loCD45RA−•VE-cadherin is a functionally important surface antigen of AGM region HSCs
Human hematopoietic stem cells (HSCs) first appear in the aorta-gonad-mesonephros (AGM) region. Medvinsky and colleagues demonstrate that the first human HSCs emerge asymmetrically in the ventral domain of the dorsal aorta (AoV) and are CD34+VE-cadherin+CD45+C-KIT+THY-1+Endoglin+RUNX1+CD38−/loCD45RA−. This study provides a foundation for further mechanistic analysis of early HSC specification during human development.
doi:10.1016/j.stemcr.2014.02.004
PMCID: PMC3986508  PMID: 24749070
17.  The kisspeptin-GnRH pathway in human reproductive health and disease 
Human Reproduction Update  2014;20(4):485-500.
BACKGROUND
The discovery of kisspeptin as key central regulator of GnRH secretion has led to a new level of understanding of the neuroendocrine regulation of human reproduction. The related discovery of the kisspeptin-neurokinin B-dynorphin (KNDy) pathway in the last decade has further strengthened our understanding of the modulation of GnRH secretion by endocrine, metabolic and environmental inputs. In this review, we summarize current understanding of the physiological roles of these novel neuropeptides, and discuss the clinical relevance of these discoveries and their potential translational applications.
METHODS
A systematic literature search was performed using PUBMED for all English language articles up to January 2014. In addition, the reference lists of all relevant original research articles and reviews were examined. This review focuses mainly on published human studies but also draws on relevant animal data.
RESULTS
Kisspeptin is a principal regulator of the secretion of gonadotrophins, and through this key role it is critical for the onset of puberty, the regulation of sex steroid-mediated feedback and the control of adult fertility. Although there is some sexual dimorphism, both neuroanatomically and functionally, these functions are apparent in both men and women. Kisspeptin acts upstream of GnRH and, following paracrine stimulatory and inhibitory inputs from neurokinin B and dynorphin (KNDy neuropeptides), signals directly to GnRH neurones to control pulsatile GnRH release. When administered to humans in different isoforms, routes and doses, kisspeptin robustly stimulates LH secretion and LH pulse frequency. Manipulation of the KNDy system is currently the focus of translational research with the possibility of future clinical application to regulate LH pulsatility, increasing gonadal sex steroid secretion in reproductive disorders characterized by decreased LH pulsatility, including hypothalamic amenorrhoea and hypogonadotropic hypogonadism. Conversely there may be scope to reduce the activity of the KNDy system to reduce LH secretion where hypersecretion of LH adds to the phenotype, such as in polycystic ovary syndrome.
CONCLUSIONS
Kisspeptin is a recently discovered neuromodulator that controls GnRH secretion mediating endocrine and metabolic inputs to the regulation of human reproduction. Manipulation of kisspeptin signalling has the potential for novel therapies in patients with pathologically low or high LH pulsatility.
doi:10.1093/humupd/dmu009
PMCID: PMC4063702  PMID: 24615662
kisspeptin; kisspeptin-neurokinin B-dynorphin; GnRH; LH pulsatility
18.  Quantitative Serial MRI of the Treated Fibroid Uterus 
PLoS ONE  2014;9(3):e89809.
Objective
There are no long-term medical treatments for uterine fibroids, and non-invasive biomarkers are needed to evaluate novel therapeutic interventions. The aim of this study was to determine whether serial dynamic contrast-enhanced MRI (DCE-MRI) and magnetization transfer MRI (MT-MRI) are able to detect changes that accompany volume reduction in patients administered GnRH analogue drugs, a treatment which is known to reduce fibroid volume and perfusion. Our secondary aim was to determine whether rapid suppression of ovarian activity by combining GnRH agonist and antagonist therapies results in faster volume reduction.
Methods
Forty women were assessed for eligibility at gynaecology clinics in the region, of whom thirty premenopausal women scheduled for hysterectomy due to symptomatic fibroids were randomized to three groups, receiving (1) GnRH agonist (Goserelin), (2) GnRH agonist+GnRH antagonist (Goserelin and Cetrorelix) or (3) no treatment. Patients were monitored by serial structural, DCE-MRI and MT-MRI, as well as by ultrasound and serum oestradiol concentration measurements from enrolment to hysterectomy (approximately 3 months).
Results
A volumetric treatment effect assessed by structural MRI occurred by day 14 of treatment (9% median reduction versus 9% increase in untreated women; P = 0.022) and persisted throughout. Reduced fibroid perfusion and permeability assessed by DCE-MRI occurred later and was demonstrable by 2–3 months (43% median reduction versus 20% increase respectively; P = 0.0093). There was no apparent treatment effect by MT-MRI. Effective suppression of oestradiol was associated with early volume reduction at days 14 (P = 0.041) and 28 (P = 0.0061).
Conclusion
DCE-MRI is sensitive to the vascular changes thought to accompany successful GnRH analogue treatment of uterine fibroids and should be considered for use in future mechanism/efficacy studies of proposed fibroid drug therapies. GnRH antagonist administration does not appear to accelerate volume reduction, though our data do support the role of oestradiol suppression in GnRH analogue treatment of fibroids.
Trial Registration
ClinicalTrials.gov NCT00746031
doi:10.1371/journal.pone.0089809
PMCID: PMC3946427  PMID: 24608161
19.  A compendium of RNA-binding motifs for decoding gene regulation 
Nature  2013;499(7457):172-177.
RNA-binding proteins are key regulators of gene expression, yet only a small fraction have been functionally characterized. Here we report a systematic analysis of the RNA motifs recognized by RNA-binding proteins, encompassing 205 distinct genes from 24 diverse eukaryotes. The sequence specificities of RNA-binding proteins display deep evolutionary conservation, and the recognition preferences for a large fraction of metazoan RNA-binding proteins can thus be inferred from their RNA-binding domain sequence. The motifs that we identify in vitro correlate well with in vivo RNA-binding data. Moreover, we can associate them with distinct functional roles in diverse types of post-transcriptional regulation, enabling new insights into the functions of RNA-binding proteins both in normal physiology and in human disease. These data provide an unprecedented overview of RNA-binding proteins and their targets, and constitute an invaluable resource for determining post-transcriptional regulatory mechanisms in eukaryotes.
doi:10.1038/nature12311
PMCID: PMC3929597  PMID: 23846655
20.  The Novel Poly(A) Polymerase Star-PAP is a Signal-Regulated Switch at the 3′-end of mRNAs 
The mRNA 3′-untranslated region (3′-UTR) modulates message stability, transport, intracellular location and translation. We have discovered a novel nuclear poly(A) polymerase termed Star-PAP (nuclear speckle targeted PIPKIα regulated-poly(A) polymerase) that couples with the transcriptional machinery and is regulated by the phosphoinositide lipid messenger phosphatidylinositol-4,5-bisphosphate (PI4,5P2), the central lipid in phosphoinositide signaling. PI4,5P2 is generated primarily by type I phosphatidylinositol phosphate kinases (PIPKI). Phosphoinositides are present in the nucleus including at nuclear speckles compartments separate from known membrane structures. PIPKs regulate cellular functions by interacting with PI4,5P2 effectors where PIPKs generate PI4,5P2 that then modulates the activity of the associated effectors. Nuclear PIPKIα interacts with and regulates Star-PAP, and PI4,5P2 specifically activates Star-PAP in a gene- and signaling-dependent manner. Importantly, other select signaling molecules integrated into the Star-PAP complex seem to regulate Star-PAP activities and processivities toward RNA substrates, and unique sequence elements around the Star-PAP binding sites within the 3′-UTR of target genes contribute to Star-PAP specificity for processing. Therefore, Star-PAP and its regulatory molecules form a signaling nexus at the 3′-end of target mRNAs to control the expression of select group of genes including the ones involved in stress responses.
doi:10.1016/j.jbior.2012.10.004
PMCID: PMC3626085  PMID: 23306079
21.  Platelet-derived growth factor receptor (PDGFR) expression in primary spinal cord gliomas 
Journal of neuro-oncology  2011;106(2):10.1007/s11060-011-0666-6.
Abnormal signaling through the platelet-derived growth factor receptor (PDGFR) has been proposed as a possible mechanism of spinal cord glioma initiation and progression. However, the extent of PDGFR expression in human spinal cord gliomas remains unknown. In this study we perform immunohistochemical analysis of PDGFRα expression in a series of 33 primary intramedullary spinal cord gliomas of different types and grades. PDGFRα was seen to be expressed in a significant subset of these tumors across all major glioma types including ependymoma, oligodendroglioma, pilocytic astrocytoma, astrocytoma, and glioblastoma. These results support the hypothesis that growth factor signaling through the PDGFR may be important for the development of at least a subset of human spinal cord gliomas. Further studies investigating the prognostic significance of PDGFR expression as well as the role of PDGF signaling on the development of intramedullary spinal cord gliomas are warranted.
doi:10.1007/s11060-011-0666-6
PMCID: PMC3869991  PMID: 21789698
Astrocytoma; Ependymoma; Glioblastoma multiforme; Glioma; Intramedullary; Oligodendroglioma; Pilocytic; Spinal cord; Tumor
22.  Sterol-Dependent Nuclear Import of ORP1S Promotes LXR Regulated Trans-Activation of APOE 
Experimental cell research  2012;318(16):10.1016/j.yexcr.2012.06.012.
Oxysterol binding protein related protein 1S (ORP1S) is a member of a family of sterol transport proteins. Here we present evidence that ORP1S translocates from the cytoplasm to the nucleus in response to sterol binding. The sterols that best promote nuclear import of ORP1S also activate the liver X receptor (LXR) transcription factors and we show that ORP1S binds to LXRs, promotes binding of LXRs to LXR response elements (LXREs) and specifically enhances LXR-dependent transcription via the ME.1 and ME.2 enhancer elements of the apoE gene. We propose that ORP1S is a cytoplasmic sterol sensor, which transports sterols to the nucleus and promotes LXR-dependent gene transcription through select enhancer elements.
doi:10.1016/j.yexcr.2012.06.012
PMCID: PMC3867128  PMID: 22728266
ORP1; ORP1S; oxysterol; LXR; nuclear import; NLS
23.  Cinnamon Counteracts the Negative Effects of a High Fat/High Fructose Diet on Behavior, Brain Insulin Signaling and Alzheimer-Associated Changes 
PLoS ONE  2013;8(12):e83243.
Insulin resistance leads to memory impairment. Cinnamon (CN) improves peripheral insulin resistance but its effects in the brain are not known. Changes in behavior, insulin signaling and Alzheimer-associated mRNA expression in the brain were measured in male Wistar rats fed a high fat/high fructose (HF/HFr) diet to induce insulin resistance, with or without CN, for 12 weeks. There was a decrease in insulin sensitivity associated with the HF/HFr diet that was reversed by CN. The CN fed rats were more active in a Y maze test than rats fed the control and HF/HFr diets. The HF/HFr diet fed rats showed greater anxiety in an elevated plus maze test that was lessened by feeding CN. The HF/HFr diet also led to a down regulation of the mRNA coding for GLUT1 and GLUT3 that was reversed by CN in the hippocampus and cortex. There were increases in Insr, Irs1 and Irs2 mRNA in the hippocampus and cortex due to the HF/HFr diet that were not reversed by CN. Increased peripheral insulin sensitivity was also associated with increased glycogen synthase in both hippocampus and cortex in the control and HF/HFr diet animals fed CN. The HF/HFr diet induced increases in mRNA associated with Alzheimers including PTEN, Tau and amyloid precursor protein (App) were also alleviated by CN. In conclusion, these data suggest that the negative effects of a HF/HFr diet on behavior, brain insulin signaling and Alzheimer-associated changes were alleviated by CN suggesting that neuroprotective effects of CN are associated with improved whole body insulin sensitivity and related changes in the brain.
doi:10.1371/journal.pone.0083243
PMCID: PMC3862724  PMID: 24349472
24.  Phenotype of Subjects with Type 2 Diabetes May Determine Clinical Response to Chromium Supplementation 
Metabolism: clinical and experimental  2007;56(12):10.1016/j.metabol.2007.07.007.
Background and Aims
Considerable controversy exists regarding use of chromium (Cr) supplementation to modulate carbohydrate metabolism in subjects with diabetes. Recently, we reported that Cr supplementation, provided as 1000 ug/day as Cr picolinate, enhanced insulin sensitivity in subjects with Type 2 diabetes. Our data agreed with some, but not all, studies that evaluated a similar dose and formulation in Type 2 diabetes and suggested that subject selection and characteristics may be important considerations when assessing the clinical response. Thus, the goal of this study was to assess which metabolic or clinical characteristics, when obtained at baseline, best determine a clinical response to Cr when assessing changes in insulin sensitivity.
Methods
Seventy-three subjects with Type 2 diabetes were assessed in a double-blinded, randomized, placebo-controlled study. Subjects were assessed at baseline for glycemic control with HbA1c measures, oral glucose tolerance tests, and body weight and body fat measures (DEXA). After baseline, insulin sensitivity in vivo was assessed with use of hyperinsulinemic-euglycemic clamps. After the baseline clamp, subjects were randomized to receive Cr supplementation (1000 ug Cr/day provided as chromium picolinate) or placebo daily for 6 months. All study parameters were repeated after 6 months. The relationship of the baseline characteristics of the study subjects to the change in insulin sensitivity was determined.
Results
63% of the subjects with Type 2 diabetes responded to the Cr treatment as compared to 30% with placebo. The only subject variable significantly associated with the clinical response to Cr was the baseline insulin sensitivity, as assessed with the hyperinsulinemic-euglycemic clamp (partial R2 = .4038) (p = .0004).
Conclusion
Subject phenotype appears to be very important when assessing the clinical response to Cr as baseline insulin sensitivity was found to account for nearly 40% of the variance in the clinical response to Cr
doi:10.1016/j.metabol.2007.07.007
PMCID: PMC3838889  PMID: 17998017
25.  Pretreatment anti-Müllerian hormone predicts for loss of ovarian function after chemotherapy for early breast cancer☆ 
European Journal of Cancer  2013;49(16):3404-3411.
Aim
Improving survival for women with early breast cancer (eBC) requires greater attention to the consequences of treatment, including risk to ovarian function. We have assessed whether biochemical markers of the ovarian reserve might improve prediction of chemotherapy related amenorrhoea.
Methods
Women (n = 59, mean age 42.6 years [(range 23.3–52.5]) with eBC were recruited before any treatment. Pretreatment ovarian reserve markers (anti-Müllerian hormone [AMH], follicle-stimulating hormone [FSH], inhibin B) were analysed in relation to ovarian status at 2 years.
Results
Pretreatment AMH was significantly lower in women with amenorrhoea at 2 years (4.0 ± 0.9 pmol/L versus 17.2 ± 2.5, P < 0.0001), but FSH and inhibin B did not differ between groups. By logistic regression, pretreatment AMH, but not age, FSH or inhibin B, was an independent predictor of ovarian status at 2 years (P = 0.005; odds ratio 0.013). We combined these data with a similar cohort (combined n = 75); receiver–operator characteristic analysis for AMH gave area under curve (AUC) of 0.90 (95% confidence interval (CI) 0.82–0.97)). A cross-validated classification tree analysis resulted in a binary classification schema with sensitivity 98.2% and specificity 80.0% for correct classification of amenorrhoea.
Conclusion
Pretreatment AMH is a useful predictor of long term post chemotherapy loss of ovarian function in women with eBC, adding significantly to the only previously established individualising predictor, i.e. age. AMH measurement may assist decision-making regarding treatment options and fertility preservation procedures.
doi:10.1016/j.ejca.2013.07.014
PMCID: PMC3807650  PMID: 23968732
AMH; Ovarian reserve; Chemotherapy; Amenorrhoea; Fertility; Breast cancer

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