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Easley C.A.,Magee Womens Research Institute and Foundation | Easley C.A.,University of Pittsburgh | Ben-Yehudah A.,Magee Womens Research Institute and Foundation | Ben-Yehudah A.,University of Pittsburgh | And 10 more authors.
Cellular Reprogramming | Year: 2010

Deciding to exit pluripotency and undergo differentiation is of singular importance for pluripotent cells, including embryonic stem cells (ESCs). The molecular mechanisms for these decisions to differentiate, as well as reversing those decisions during induced pluripotency (iPS), have focused largely on transcriptomic controls. Here, we explore the role of translational control for the maintenance of pluripotency and the decisions to differentiate. Global protein translation is significantly reduced in hESCs compared to their differentiated progeny. Furthermore, p70 S6K activation is restricted in hESCs compared to differentiated fibroblast-like cells. Disruption of p70 S6K-mediated translation by rapamycin or siRNA knockdown in undifferentiated hESCs does not alter cell viability or expression of the pluripotency markers Oct4 and Nanog. However, expression of constitutively active p70 S6K, but not wild-type p70 S6K, induces differentiation. Additionally, hESCs exhibit high levels of the mTORC1/p70 S6K inhibitory complex TSC1/TSC2 and preferentially express more rapamycin insensitive mTORC2 compared to differentiated cells. siRNA-mediated knockdown of both TSC2 and Rictor elevates p70 S6K activation and induces differentiation of hESCs. These results suggest that hESCs tightly regulate mTORC1/p70 S6K-mediated protein translation to maintain a pluripotent state as well as implicate a novel role for protein synthesis as a driving force behind hESC differentiation. Copyright 2010, Mary Ann Liebert, Inc.


Oliphant S.S.,University of Pittsburgh | Oliphant S.S.,University of Arkansas for Medical Sciences | Bochenska K.,University of Pittsburgh | Tolge M.E.,Magee Womens Research Institute and Foundation | And 2 more authors.
International Urogynecology Journal and Pelvic Floor Dysfunction | Year: 2014

Introduction and hypothesis: To determine the incidence of lower urinary tract (LUT) injury at the time of Cesarean delivery (CD) and to identify factors associated with LUT injury.Methods: Cases of LUT injury at delivery between 2001 and 2012, were identified by ICD-9 code. Chart review was utilized for verification and descriptive data collection. LUT injury incidence rates were calculated using annual delivery totals and trends over time were calculated using simple linear regression. LUT injury was classified as full-thickness bladder injury (including ureteral injury) or partial-thickness bladder injury based on degree of injury and post-operative intervention. Each case was year-matched to generate two CD controls. Logistic regression analysis was performed using maternal, delivery, and health system characteristics to identify factors associated with full or partial injury. Appropriate statistical analyses were performed with significance at p < 0.05.Results: Overall delivery and CD rates increased during the study time period, but despite the increase in CD rates, annual rates of LUT injury did not vary significantly (p = 0.658). Of the 72 LUT injuries reported, 39 (54 %) were full-thickness bladder, 2 (3 %) ureteral, and 31 (43 %) were partial-thickness bladder injuries. Full injury, controlling for repeat CD, was associated with increasing maternal age, transfusion, and active second stage of labor. Partial injury, was associated with increasing maternal age and delivery in the first half of the academic year.Conclusions: Despite an increasing volume of CDs, LUT injury remained relatively uncommon (0.3 % of all CDs). Full and partial bladder injuries have unique risk profiles. © 2014, The International Urogynecological Association.


Ben-Yehudah A.,University of Pittsburgh | Ben-Yehudah A.,Magee Womens Research Institute and Foundation | Campanaro B.M.,University of Pittsburgh | Wakefield L.M.,University of Pittsburgh | And 7 more authors.
Respiratory Research | Year: 2013

Background: The ability of chemicals to disrupt neonatal development can be studied using embryonic stem cells (ESC). One such chemical is nicotine. Prenatal nicotine exposure is known to affect postnatal lung function, although the mechanisms by which it has this effect are not clear. Since fibroblasts are a critical component of the developing lung, providing structure and secreting paracrine factors that are essential to epithelialization, this study focuses on the differentiation of ESC into fibroblasts using a directed differentiation protocol.Methods: Fibroblasts obtained from non-human primate ESC (nhpESC) differentiation were analyzed by immunohistochemistry, immunostaining, Affymetrix gene expression array, qPCR, and immunoblotting.Results: Results of these analyses demonstrated that although nhpESCs differentiate into fibroblasts in the presence of nicotine and appear normal by some measures, including H&E and SMA staining, they have an altered gene expression profile. Network analysis of expression changes demonstrated an over-representation of cell-cycle related genes with downregulation of N-myc as a central regulator in the pathway. Further investigation demonstrated that cells differentiated in the presence of nicotine had decreased N-myc mRNA and protein expression and longer doubling times, a biological effect consistent with downregulation of N-myc.Conclusions: This study is the first to use primate ESC to demonstrate that nicotine can affect cellular differentiation from pluripotency into fibroblasts, and in particular, mediate N-myc expression in differentiating ESCs. Given the crucial role of fibroblasts throughout the body, this has important implications for the effect of cigarette smoke exposure on human development not only in the lung, but in organogenesis in general. © 2013 Ben-Yehudah et al.; licensee BioMed Central Ltd.


Hoffner L.,Magee Womens Research Institute and Foundation | Surti U.,Magee Womens Research Institute and Foundation | Surti U.,University of Pittsburgh
Cancer Genetics | Year: 2012

Gestational choriocarcinoma is usually a rapidly spreading fatal disease, but it is curable if diagnosed early and treated. It is a unique malignancy that is a partial or complete allograft with a genotype that is not the same as the host genotype. It is most often preceded by an abnormal molar pregnancy. The surprising and unique androgenetic origin of complete hydatidiform molar pregnancies was first revealed by Kajii and Ohama in 1977. We describe the current understanding of the morphology, epidemiology and genetics of gestational trophoblastic disease that followed the milestone findings by Kajii and Ohama. © 2012 Elsevier Inc.


Easley IV C.A.,University of Pittsburgh | Easley IV C.A.,Magee Womens Research Institute and Foundation | Miki T.,McGowan Institute for Regenerative Medicine | Castro C.A.,Magee Womens Research Institute and Foundation | And 6 more authors.
Cellular Reprogramming | Year: 2012

Cellular reprogramming from adult somatic cells into an embryonic cell-like state, termed induced pluripotency, has been achieved in several cell types. However, the ability to reprogram human amniotic epithelial cells (hAECs), an abundant cell source derived from discarded placental tissue, has only recently been investigated. Here we show that not only are hAECs easily reprogrammed into induced pluripotent stem cells (AE-iPSCs), but hAECs reprogram faster and more efficiently than adult and neonatal somatic dermal fibroblasts. Furthermore, AE-iPSCs express higher levels of NANOG and OCT4 compared to human foreskin fibroblast iPSCs (HFF1-iPSCs) and express decreased levels of genes associated with differentiation, including NEUROD1 and SOX17, markers of neuronal differentiation. To elucidate the mechanism behind the higher reprogramming efficiency of hAECs, we analyzed global DNA methylation, global histone acetylation, and the mitochondrial DNA A3243G point mutation. Whereas hAECs show no differences in global histone acetylation or mitochondrial point mutation accumulation compared to adult and neonatal dermal fibroblasts, hAECs demonstrate a decreased global DNA methylation compared to dermal fibroblasts. Likewise, quantitative gene expression analyses show that hAECs endogenously express OCT4, SOX2, KLF4, and c-MYC, all four factors used in cellular reprogramming. Thus, hAECs represent an ideal cell type for testing novel approaches for generating clinically viable iPSCs and offer significant advantages over postnatal cells that more likely may be contaminated by environmental exposures and infectious agents. © Mary Ann Liebert, Inc.


Bell M.J.,University of Pittsburgh | Bell M.J.,Magee Womens Research Institute and Foundation | Roberts J.M.,Magee Womens Research Institute and Foundation | Roberts J.M.,University of Pittsburgh | And 6 more authors.
BMC Pregnancy and Childbirth | Year: 2013

Background: Preeclampsia is a hypertensive, multi-system pregnancy disorder whose pathophysiology remains unclear. Elevations in circulating soluble endoglin (sENG) and placental/blood ENG mRNA expression antedate the clinical onset of preeclampsia. This study investigated if endoglin (ENG) pathway genetic variation was also associated with the development of preeclampsia.Methods: We used a case-control candidate gene association design. Data from 355 white (181 preeclampsia cases/174 controls) and 60 black (30 preeclampsia cases/30 controls) women matched on ancestry, age, and parity were analyzed. Tagging single nucleotide polymorphisms (tSNPs) and potentially functional SNPs in ENG, TGFβ1, TGFβR1, ALK1, and TGFβR2 were genotyped with iPLEX® and TaqMan®. Chi-square or Fisher's exact tests were used to conduct allele/genotype/haplotype tests in white/black subgroups separately. Odds ratios were computed with binary logistic regression for tSNPs with significant genotype tests.Results: Of the 49 SNPs evaluated, variation in two ENG tSNPs (rs11792480, rs10121110) and one TGFβR2 tSNP (rs6550005) was associated with preeclampsia in white women (P <0.05, each). In black women, variation in two TGFβ1 tSNPs (rs4803455, rs4803457), one TGFβR1 tSNP (rs10739778), and three TGFβR2 tSNPs (rs6550005, rs1346907, rs877572) was associated with preeclampsia (P <0.05, each). Further evaluation of ENG tSNP rs10121110 revealed that white women inheriting the AA genotype were 2.29 times more likely to develop preeclampsia compared to the GG genotype (P = 0.008, [99% CI: 1.02 to 5.13]). For black women, similar evaluation of TGFβ1 tSNP rs4803457 revealed women inheriting the CT genotype were 7.44 times more likely to develop preeclampsia than those with the CC genotype (P = 0.005, [99% CI: 1.19 to 46.41]).Conclusions: ENG pathway genetic variation is associated with preeclampsia. Different ENG pathway genes may be involved in preeclampsia development among white and black women. Additional studies are needed to validate these findings and to determine if genetic variation in ENG pathway genes impacts ENG and sENG levels in preeclampsia. © 2013 Bell et al.; licensee BioMed Central Ltd.


PubMed | Magee Womens Research Institute and Foundation
Type: Journal Article | Journal: Cancer genetics | Year: 2012

Gestational choriocarcinoma is usually a rapidly spreading fatal disease, but it is curable if diagnosed early and treated. It is a unique malignancy that is a partial or complete allograft with a genotype that is not the same as the host genotype. It is most often preceded by an abnormal molar pregnancy. The surprising and unique androgenetic origin of complete hydatidiform molar pregnancies was first revealed by Kajii and Ohama in 1977. We describe the current understanding of the morphology, epidemiology and genetics of gestational trophoblastic disease that followed the milestone findings by Kajii and Ohama.


PubMed | Magee Womens Research Institute and Foundation
Type: Journal Article | Journal: Cellular reprogramming | Year: 2010

Deciding to exit pluripotency and undergo differentiation is of singular importance for pluripotent cells, including embryonic stem cells (ESCs). The molecular mechanisms for these decisions to differentiate, as well as reversing those decisions during induced pluripotency (iPS), have focused largely on transcriptomic controls. Here, we explore the role of translational control for the maintenance of pluripotency and the decisions to differentiate. Global protein translation is significantly reduced in hESCs compared to their differentiated progeny. Furthermore, p70 S6K activation is restricted in hESCs compared to differentiated fibroblast-like cells. Disruption of p70 S6K-mediated translation by rapamycin or siRNA knockdown in undifferentiated hESCs does not alter cell viability or expression of the pluripotency markers Oct4 and Nanog. However, expression of constitutively active p70 S6K, but not wild-type p70 S6K, induces differentiation. Additionally, hESCs exhibit high levels of the mTORC1/p70 S6K inhibitory complex TSC1/TSC2 and preferentially express more rapamycin insensitive mTORC2 compared to differentiated cells. siRNA-mediated knockdown of both TSC2 and Rictor elevates p70 S6K activation and induces differentiation of hESCs. These results suggest that hESCs tightly regulate mTORC1/p70 S6K-mediated protein translation to maintain a pluripotent state as well as implicate a novel role for protein synthesis as a driving force behind hESC differentiation.


PubMed | Magee Womens Research Institute and Foundation
Type: Journal Article | Journal: Journal of clinical microbiology | Year: 2011

Streptococcus pseudoporcinus, a beta-hemolytic microorganism first isolated from the female gastrourinary tract in 2006, cross-reacts with serogrouping kits for group B Streptococcus (GBS) and could be misidentified in the laboratory. The epidemiologic characteristics of this species have not been reported previously, but this organism is thought to be rare. Paired vaginal and rectal samples were collected from 663 nonpregnant women enrolled in a phase II clinical vaccine trial of a GBS type III capsular polysaccharide-protein conjugate vaccine, and isolates initially identified as S. pseudoporcinus were collected for further testing. A total of 120 isolates of S. pseudoporcinus were recovered from 36 unique individuals with 5.4% of 663 women having this organism recovered at least once during follow-up. All of these isolates cross-reacted with a commercially available GBS serogrouping kit. Women colonized with isolates confirmed as S. pseudoporcinus by genotypic and phenotypic methodologies were compared to women who were not colonized to determine whether there were any significant factors associated with acquisition of S. pseudoporcinus. Acquisition of S. pseudoporcinus vaginally and/or rectally was 36 per 846.0 women-years of follow-up for an annual incidence of 4 per 100 woman-years of follow-up. Acquisition of S. pseudoporcinus was independently associated with black women, being 30 to 40 years of age, recent Trichomonas vaginalis infection, primary or recurrent genital herpes, having bacterial vaginosis by Nugent criteria, and having had two or more male sexual partners since the last visit. This study suggests that S. pseudoporcinus is not rare, especially among black women, and could be misidentified as GBS.

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