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1.  The impact of novel retinoids in combination with platinum chemotherapy on ovarian cancer stem cells 
Gynecologic oncology  2011;125(1):226-230.
Retinoids are important modulators of cell growth, differentiation, and proliferation. 9cUAB30, 9cUAB124, and 9cUAB130 are three novel retinoid compounds that show cytotoxic effects in other malignancies. We evaluated these novel retinoids in combination with chemotherapy against ovarian cancer stem cells (CSCs) in vitro and in an ex vivo model.
A2780 cells were plated in 96-well plates and treated with retinoid, carboplatin, or combination therapy. Cell viability was evaluated using ATPLite assay. The A2780 cell line was also analyzed for CSCs by evaluating ALDH activity using flow cytometry. A2780 cells treated ex vivo with retinoids and chemotherapy were injected into the flank of athymic nude mice in order to evaluate subsequent tumor initiating capacity.
A2780 cells were sensitive to treatment with retinoids and carboplatin. The best treatment resulted from the combination of retinoid 9cUAB130 and carboplatin. Untreated A2780 cells demonstrated ALDH activity in 3.3% of the cell population. Carboplatin treatment enriched ALDH activity to 27.3%, while 9cUAB130±carboplatin maintained the ALDH positive levels similar to untreated controls (2.3% and 6.7%, respectively). Similar results were found in tumorsphere-forming conditions. Flank injections of ex vivo treated A2780 cells resulted in 4/4 mice developing tumors at 40 days in the untreated group, while 0/4 tumors developed in the 9cUAB130 and carboplatin treated group.
Combination treatment with carboplatin and retinoids reduced cell-viability, reduced CSC marker expression, and inhibited tumorigenicity, making it a more effective treatment when compared with carboplatin alone.
PMCID: PMC4049085  PMID: 22155260
Retinoid; Ovarian cancer; Stem cell
2.  Constructing a Novel Simple LEEP Training Model 
Duty hour restrictions and enhanced focus on patient safety have prompted the development of new instruction models for practice of surgical techniques outside the operating room, including models for teaching loop electrosurgical excisional procedure (LEEP), a common procedure that gynecology residents perform to diagnose and manage cervical disease.
We sought to develop an inexpensive and reusable training model for guided practice opportunities that will improve gynecology residents' LEEP technique.
Polyvinyl chloride, foam, and a polish sausage are used to simulate the basic anatomy of the vagina and cervix. A 2-in-diameter polyvinyl chloride pipe and high-density foam are used to create a realistic representation with the sausage simulating the cervix. An electrosurgical pad is attached to the sausage and a standard operating room electrosurgical generator is used.
After a brief lecture and demonstration of the LEEP procedure, gynecology residents are positioned at individual stations. Use of 2 to 3 instructors allows for the provision of directions and feedback to residents as they perform the simulated LEEP. During the last 6 years, this model has continued to improve residents' confidence and skills with the procedure.
An anatomically accurate LEEP model can not only improve resident knowledge, skills, and confidence, but also improve quality and patient safety. This training model allows residents to refine their surgical skills through guided practice and instructors to monitor performance before residents to perform the procedure on patients.
PMCID: PMC3693702  PMID: 24404281
3.  Predictors of Plasma DDT and DDE Concentrations among Women Exposed to Indoor Residual Spraying for Malaria Control in the South African Study of Women and Babies (SOWB) 
Environmental Health Perspectives  2014;122(6):545-552.
Background: Few studies have examined predictors of DDT (dichlorodiphenyltrichloroethane) and DDE (dichlorodiphenyldichloroethylene) levels among residents in homes sprayed with DDT for malaria control with the aim of identifying exposure-reduction strategies.
Methods: The present analysis included 381 women enrolled in the Study of Women and Babies (SOWB) during 2010–2011, from eight South African villages in the Limpopo Province, South Africa. Indoor residual spraying (IRS) occurred in half of the villages. Questionnaires regarding various demographic and medical factors were administered and blood samples were obtained. We classified the women into three exposure groups by type of residence: unsprayed village (n = 175), IRS village in household with a low likelihood of DDT use (non-DDT IRS household, n = 106), IRS village in household with a high likelihood of DDT use (DDT IRS household, n = 100). We used multivariable models of natural log-transformed DDT plasma levels (in micrograms per liter) and DDE (in micrograms per liter) to identify predictors for each group.
Results: Median levels of DDT and DDE among women in unsprayed villages were 0.3 [interquartile range (IQR): 0.1–0.9] and 1.7 (IQR: 0.7–5.5), respectively. Median levels of DDT and DDE among women in DDT IRS households were 2.6 (IQR: 1.1–6.6) and 8.5 (IQR: 4.7–18.0), respectively. In unsprayed villages, women with water piped to the yard, rather than a public tap, had 73% lower DDT (95% CI: –83, –57%) and 61% lower DDE (95% CI: –74, –40%) levels. In DDT IRS households, women who reported taking more than six actions to prepare their home before IRS (e.g., covering water and food) had 40% lower DDT levels (95% CI: –63, –0.3%) than women who took fewer than four actions.
Conclusion: The predictors of DDT and DDE plasma levels identified in the present study may inform interventions aimed at decreasing exposure. Among households where DDT is likely to be used for IRS, education regarding home preparations may provide an interventional target.
Citation: Whitworth KW, Bornman RM, Archer JI, Kudumu MO, Travlos GS, Wilson RE, Longnecker MP. 2014. Predictors of plasma DDT and DDE concentrations among women exposed to indoor residual spraying for malaria control in the South African Study of Women and Babies (SOWB). Environ Health Perspect 122:545–552;
PMCID: PMC4048257  PMID: 24577839
4.  Dickkopf-Related Protein 1 Inhibits the WNT Signaling Pathway and Improves Pig Oocyte Maturation 
PLoS ONE  2014;9(4):e95114.
The ability to mature oocytes in vitro provides a tool for creating embryos by parthenogenesis, fertilization, and cloning. Unfortunately the quality of oocytes matured in vitro falls behind that of in vivo matured oocytes. To address this difference, transcriptional profiling by deep sequencing was conducted on pig oocytes that were either matured in vitro or in vivo. Alignment of over 18 million reads identified 1,316 transcripts that were differentially represented. One pathway that was overrepresented in the oocytes matured in vitro was for Wingless-type MMTV integration site (WNT) signaling. In an attempt to inhibit the WNT pathway, Dickkopf-related protein 1 was added to the in vitro maturation medium. Addition of Dickkopf-related protein 1 improved the percentage of oocytes that matured to the metaphase II stage, increased the number of nuclei in the resulting blastocyst stage embryos, and reduced the amount of disheveled segment polarity protein 1 protein in oocytes. It is concluded that transcriptional profiling is a powerful method for detecting differences between in vitro and in vivo matured oocytes, and that the WNT signaling pathway is important for proper oocyte maturation.
PMCID: PMC3989281  PMID: 24739947
5.  Determinants of plasma concentrations of perfluoroalkyl substances in pregnant Norwegian women 
Environment international  2013;54:74-84.
Perfluoroalkyl substances (PFASs) are widespread pollutants that have been associated with adverse health effects although not on a consistent basis. Diet has been considered the main source of exposure. The aim of the present study was to identify determinants of four plasma PFASs in pregnant Norwegian women.
This study is based in the Norwegian Mother and Child Cohort Study (MoBa) conducted by the Norwegian Institute of Public Health. Our sample included 487 women who enrolled in MoBa from 2003–2004. A questionnaire regarding sociodemographic, medical, and reproductive history was completed at 17 weeks gestation and a dietary questionnaire was completed at 22 weeks gestation. Maternal plasma samples were obtained around 17 weeks of gestation. Plasma concentrations of four PFASs (perfluorooctane sulfonate (PFOS), perfluorooctanoate (PFOA), perfluorohexane sulfonate (PFHxS), and perfluorononanoate (PFNA)) were examined in relation to demographic, lifestyle, dietary, and pregnancy-related covariates. Predictors were identified by optimizing multiple linear regression models using Akaike’s information criterion (AIC).
Parity was the determinant with the largest influence on plasma PFAS concentrations, with r2 between 0.09 and 0.32 in simple regression models. In optimal multivariate models, when compared to nulliparous women, parous women had 46%, 70%, 19%, and 62% lower concentrations of PFOS, PFOA, PFHxS, and PFNA respectively (p<0.001 except for PFHxS, p<0.01). In all these models, duration of breastfeeding was associated with reduced PFAS levels. PFOA showed the largest reduction from breastfeeding, with a 2–3% reduction per month of breastfeeding in typical cases. Levels of PFOS, PFOA, and PFNA increased with time since most recent pregnancy. While pregnancy-related factors were the most important predictors, diet was a significant factor explaining up to 4% of the variance. One quartile increase in estimated dietary PFAS intake was associated with plasma PFOS, PFOA, PFHxS, and PFNA concentration increases of 7.2%, 3.3%, 5.8% and 9.8%, respectively, resulting in small, although non-trivial absolute changes in PFAS concentrations.
The history of previous pregnancies and breastfeeding were the most important determinants of PFASs in this sample of pregnant women.
PMCID: PMC3605228  PMID: 23419425
Perfluoroalkyl substances; reproductive history; pregnancy; dietary exposure
6.  Identification of fatty acid binding protein 4 as an adipokine that regulates insulin secretion during obesity 
Molecular Metabolism  2014;3(4):465-473.
A critical feature of obesity is enhanced insulin secretion from pancreatic β-cells, enabling the majority of individuals to maintain glycaemic control despite adiposity and insulin resistance. Surprisingly, the factors coordinating this adaptive β-cell response with adiposity have not been delineated. Here we show that fatty acid binding protein 4 (FABP4/aP2) is an adipokine released from adipocytes under obesogenic conditions, such as hypoxia, to augment insulin secretion. The insulinotropic action of FABP4 was identified using an in vitro system that recapitulates adipocyte to β-cell endocrine signalling, with glucose-stimulated insulin secretion (GSIS) as a functional readout, coupled with quantitative proteomics. Exogenous FABP4 potentiated GSIS in vitro and in vivo, and circulating FABP4 levels correlated with GSIS in humans. Insulin inhibited FABP4 release from adipocytes in vitro, in mice and in humans, consistent with feedback regulation. These data suggest that FABP4 and insulin form an endocrine loop coordinating the β-cell response to obesity.
Graphical abstract
PMCID: PMC4060222  PMID: 24944906
Obesity; Adipokine; Adipocyte; Beta-cell; Insulin secretion; FABP4; T2D, type 2 diabetes; NEFA, non-esterified fatty acid; GSIS, glucose-stimulated insulin secretion; SILAC, stable-isotope labelling by amino acids in cell culture; ELISA, enzyme-linked immunosorbant assay; BMI, body mass index; cAMP, cyclic-AMP; IBMX, 3-Isobutyl-1-methylxanthine
7.  Tuberculin Skin Tests versus Interferon-Gamma Release Assays in Tuberculosis Screening among Immigrant Visa Applicants 
Objective. Use of tuberculin skin tests (TSTs) and interferon gamma release assays (IGRAs) as part of tuberculosis (TB) screening among immigrants from high TB-burden countries has not been fully evaluated. Methods. Prevalence of Mycobacterium tuberculosis infection (MTBI) based on TST, or the QuantiFERON-TB Gold test (QFT-G), was determined among immigrant applicants in Vietnam bound for the United States (US); factors associated with test results and discordance were assessed; predictive values of TST and QFT-G for identifying chest radiographs (CXRs) consistent with TB were calculated. Results. Of 1,246 immigrant visa applicants studied, 57.9% were TST positive, 28.3% were QFT-G positive, and test agreement was 59.4%. Increasing age was associated with positive TST results, positive QFT-G results, TST-positive but QFT-G-negative discordance, and abnormal CXRs consistent with TB. Positive predictive values of TST and QFT-G for an abnormal CXR were 25.9% and 25.6%, respectively. Conclusion. The estimated prevalence of MTBI among US-bound visa applicants in Vietnam based on TST was twice that based on QFT-G, and 14 times higher than a TST-based estimate of MTBI prevalence reported for the general US population in 2000. QFT-G was not better than TST at predicting abnormal CXRs consistent with TB.
PMCID: PMC3967820  PMID: 24738031
8.  An Intact Sialoadhesin (Sn/SIGLEC1/CD169) Is Not Required for Attachment/Internalization of the Porcine Reproductive and Respiratory Syndrome Virus 
Journal of Virology  2013;87(17):9538-9546.
Surface expression of SIGLEC1, also known as sialoadhesin or CD169, is considered a primary determinant of the permissiveness of porcine alveolar macrophages for infection by porcine reproductive and respiratory syndrome virus (PRRSV). In vitro, the attachment and internalization of PRRSV are dependent on the interaction between sialic acid on the virion surface and the sialic acid binding domain of the SIGLEC1 gene. To test the role of SIGLEC1 in PRRSV infection, a SIGLEC1 gene knockout pig was created by removing part of exon 1 and all of exons 2 and 3 of the SIGLEC1 gene. The resulting knockout ablated SIGLEC1 expression on the surface of alveolar macrophages but had no effect on the expression of CD163, a coreceptor for PRRSV. After infection, PRRSV viremia in SIGLEC1−/− pigs followed the same course as in SIGLEC1−/+ and SIGLEC1+/+ littermates. The absence of SIGLEC1 had no measurable effect on other aspects of PRRSV infection, including clinical disease course and histopathology. The results demonstrate that the expression of the SIGLEC1 gene is not required for infection of pigs with PRRSV and that the absence of SIGLEC1 does not contribute to the pathogenesis of acute disease.
PMCID: PMC3754101  PMID: 23785195
9.  The Parkinson’s disease genes Fbxo7 and Parkin interact to mediate mitophagy 
Nature neuroscience  2013;16(9):10.1038/nn.3489.
Compelling evidence indicates that two autosomal recessive Parkinson’s disease genes, PINK1 (PARK6) and Parkin (PARK2), co-operate to mediate the autophagic clearance of damaged mitochondria (mitophagy). Mutations in the F-box domain containing protein Fbxo7 (PARK15) also cause early onset autosomal recessive Parkinson’s disease by an unknown mechanism. Here we show that Fbxo7 participates in mitochondrial maintenance through direct interaction with PINK1 and Parkin and plays a role in Parkin-mediated mitophagy. Cells with reduced Fbxo7 expression show deficiencies in Parkin mitochondrial translocation, ubiquitination of mitofusin 1 and mitophagy. In Drosophila, ectopic overexpression of Fbxo7 rescued loss of Parkin supporting a functional relationship between the two proteins. Parkinson’s disease-causing mutations in Fbxo7 interfere with this process, emphasising the importance of mitochondrial dysfunction in Parkinson’s disease pathogenesis.
PMCID: PMC3827746  PMID: 23933751
Fbxo7; Parkin; PINK1; mitofusin 1; mitophagy; Drosophila; Parkinson’s disease
10.  High density lipoproteins improve insulin sensitivity in high-fat diet-fed mice by suppressing hepatic inflammation[S] 
Journal of Lipid Research  2014;55(3):421-430.
Obesity-induced liver inflammation can drive insulin resistance. HDL has anti-inflammatory properties, so we hypothesized that low levels of HDL would perpetuate inflammatory responses in the liver and that HDL treatment would suppress liver inflammation and insulin resistance. The aim of this study was to investigate the effects of lipid-free apoAI on hepatic inflammation and insulin resistance in mice. We also investigated apoAI as a component of reconstituted HDLs (rHDLs) in hepatocytes to confirm results we observed in vivo. To test our hypothesis, C57BL/6 mice were fed a high-fat diet (HFD) for 16 weeks and administered either saline or lipid-free apoAI. Injections of lipid-free apoAI twice a week for 2 or 4 weeks with lipid-free apoAI resulted in: i) improved insulin sensitivity associated with decreased systemic and hepatic inflammation; ii) suppression of hepatic mRNA expression for key transcriptional regulators of lipogenic gene expression; and iii) suppression of nuclear factor κB (NF-κB) activation. Human hepatoma HuH-7 cells exposed to rHDLs showed suppressed TNFα-induced NF-κB activation, correlating with decreased NF-κB target gene expression. We conclude that apoAI suppresses liver inflammation in HFD mice and improves insulin resistance via a mechanism that involves a downregulation of NF-κB activation.
PMCID: PMC3934727  PMID: 24347528
insulin resistance; apolipoprotein AI; cellular signalling
11.  The Role of Outcome Expectations in the Generation of the Feedback-related Negativity 
Psychophysiology  2012;50(2):125-133.
The Feedback-related Negativity (FRN) is thought to index activity within the midbrain dopaminergic reward-learning system, with larger FRN magnitudes observed when outcomes are worse than expected. This view holds that the FRN is an index of neural activity coding for prediction errors, and reflects activity that can be used to adaptively alter future performance. Untested to date, however, is a key prediction of this view: the FRN should not appear in response to negative outcomes when outcome expectations are not allowed to develop. The current study tests this assumption by eliciting FRNs to win and loss feedback in conditions of participant choice, participant observation of computer choice, and critically, simple presentation of win or loss feedback in the absence of a predictive choice cue. Whereas FRNs were observed in each of the conditions in which there was time for an expectation to develop, no FRN was observed in conditions without sufficient time for the development of an expectation. These results provide empirical support for an untested but central tenet of the reinforcement learning account of the genesis of the FRN.
PMCID: PMC3540152  PMID: 23153354
12.  Variability of the QuantiFERON®-TB Gold In-Tube Test Using Automated and Manual Methods 
PLoS ONE  2014;9(1):e86721.
The QuantiFERON®-TB Gold In-Tube test (QFT-GIT) detects Mycobacterium tuberculosis (Mtb) infection by measuring release of interferon gamma (IFN-γ) when T-cells (in heparinized whole blood) are stimulated with specific Mtb antigens. The amount of IFN-γ is determined by enzyme-linked immunosorbent assay (ELISA). Automation of the ELISA method may reduce variability. To assess the impact of ELISA automation, we compared QFT-GIT results and variability when ELISAs were performed manually and with automation.
Blood was collected into two sets of QFT-GIT tubes and processed at the same time. For each set, IFN-γ was measured in automated and manual ELISAs. Variability in interpretations and IFN-γ measurements was assessed between automated (A1 vs. A2) and manual (M1 vs. M2) ELISAs. Variability in IFN-γ measurements was also assessed on separate groups stratified by the mean of the four ELISAs.
Subjects (N = 146) had two automated and two manual ELISAs completed. Overall, interpretations were discordant for 16 (11%) subjects. Excluding one subject with indeterminate results, 7 (4.8%) subjects had discordant automated interpretations and 10 (6.9%) subjects had discordant manual interpretations (p = 0.17). Quantitative variability was not uniform; within-subject variability was greater with higher IFN-γ measurements and with manual ELISAs. For subjects with mean TB Responses ±0.25 IU/mL of the 0.35 IU/mL cutoff, the within-subject standard deviation for two manual tests was 0.27 (CI95 = 0.22–0.37) IU/mL vs. 0.09 (CI95 = 0.07–0.12) IU/mL for two automated tests.
QFT-GIT ELISA automation may reduce variability near the test cutoff. Methodological differences should be considered when interpreting and using IFN-γ release assays (IGRAs).
PMCID: PMC3900587  PMID: 24466211
13.  Assessing the Association between Oral Hygiene and Preterm Birth by Quantitative Light-Induced Fluorescence 
The Scientific World Journal  2014;2014:374694.
The aim of this study was to investigate the purported link between oral hygiene and preterm birth by using image analysis tools to quantify dental plaque biofilm. Volunteers (n = 91) attending an antenatal clinic were identified as those considered to be “at high risk” of preterm delivery (i.e., a previous history of idiopathic preterm delivery, case group) or those who were not considered to be at risk (control group). The women had images of their anterior teeth captured using quantitative light-induced fluorescence (QLF). These images were analysed to calculate the amount of red fluorescent plaque (ΔR%) and percentage of plaque coverage. QLF showed little difference in ΔR% between the two groups, 65.00% case versus 68.70% control, whereas there was 19.29% difference with regard to the mean plaque coverage, 25.50% case versus 20.58% control. A logistic regression model showed a significant association between plaque coverage and case/control status (P = 0.031), controlling for other potential predictor variables, namely, smoking status, maternal age, and body mass index (BMI).
PMCID: PMC3913385  PMID: 24511282
14.  Complex gene expression in the dragline silk producing glands of the Western black widow (Latrodectus hesperus) 
BMC Genomics  2013;14:846.
Orb-web and cob-web weaving spiders spin dragline silk fibers that are among the strongest materials known. Draglines are primarily composed of MaSp1 and MaSp2, two spidroins (spider fibrous proteins) expressed in the major ampullate (MA) silk glands. Prior genetic studies of dragline silk have focused mostly on determining the sequence of these spidroins, leaving other genetic aspects of silk synthesis largely uncharacterized.
Here, we used deep sequencing to profile gene expression patterns in the Western black widow, Latrodectus hesperus. We sequenced millions of 3′-anchored “tags” of cDNAs derived either from MA glands or control tissue (cephalothorax) mRNAs, then associated the tags with genes by compiling a reference database from our newly constructed normalized L. hesperus cDNA library and published L. hesperus sequences. We were able to determine transcript abundance and alternative polyadenylation of each of three loci encoding MaSp1. The ratio of MaSp1:MaSp2 transcripts varied between individuals, but on average was similar to the estimated ratio of MaSp1:MaSp2 in dragline fibers. We also identified transcription of TuSp1 in MA glands, another spidroin family member that encodes the primary component of egg-sac silk, synthesized in tubuliform glands. In addition to the spidroin paralogs, we identified 30 genes that are more abundantly represented in MA glands than cephalothoraxes and represent new candidates for involvement in spider silk synthesis.
Modulating expression rates of MaSp1 variants as well as MaSp2 and TuSp1 could lead to differences in mechanical properties of dragline fibers. Many of the newly identified candidate genes likely encode secreted proteins, suggesting they could be incorporated into dragline fibers or assist in protein processing and fiber assembly. Our results demonstrate previously unrecognized transcript complexity in spider silk glands.
PMCID: PMC3879032  PMID: 24295234
Spidroin; Major ampullate glands; Alternative polyadenylation; MaSp1; Spider; Tag profiling
15.  Biochemical evidence for an alternate pathway in N-linked glycoprotein biosynthesis 
Nature chemical biology  2013;9(6):10.1038/nchembio.1249.
Asparagine-linked glycosylation is a complex protein modification conserved among all three domains of life. Herein we report the in vitro analysis of N-linked glycosylation from the methanogenic archaeon Methanococcus voltae. Using a suite of synthetic and semisynthetic substrates, we show that AglK initiates N-linked glycosylation in M. voltae through the formation of α-linked dolichyl monophosphate N-acetylglucosamine (Dol-P-GlcNAc), which contrasts with the polyprenyl-diphosphate intermediates that feature in both eukaryotes and bacteria. Intriguingly, AglK exhibits high sequence homology to dolichyl-phosphate β-glucosyltransferases, including Alg5 in eukaryotes, suggesting a common evolutionary origin. The combined action of the first two enzymes, AglK and AglC, afforded an α-linked Dol-P-glycan that serves as a competent substrate for the archaeal oligosaccharyl transferase AglB. These studies provide the first biochemical evidence revealing that despite the apparent similarity of the overall pathways, there are actually two general strategies to achieve N-linked glycoproteins across the domains of life.
PMCID: PMC3661703  PMID: 23624439
16.  Effects of Combined Treatment of MG132 and Scriptaid on Early and Term Development of Porcine Somatic Cell Nuclear Transfer Embryos 
Cellular Reprogramming  2012;14(5):385-389.
Although improving, the efficiency of producing offspring by somatic cell nuclear transfer (SCNT) is still low (<1.5%). Our laboratory has demonstrated that histone deacetylase inhibitor (Scriptaid) treatment of reconstructed embryos enhances blastocyst formation and cloning efficiency in pigs. It has also been shown that proteasomal inhibitor MG132 treatment for 2 h after activation of oocytes increases blastocyst rate and pregnancy rate. The current experiment was carried out to determine the effects of combined MG132 and Scriptaid treatment on early embryo development in vitro and on term development in vivo. Immediately after electrofusion and activation, SCNT oocytes were treated with 0, 1, or 10 μM MG132 for 2 h in the presence of 500 nM Scriptaid, washed and treated with Scriptaid for an additional 14 to 15 h, then cultured in porcine zygote medium 3 (PZM3) until day 6. There was no difference in percent cleavage (58.1±7.2%, 62.7±7.2%, and 62.5±7.2%) on day 2, or total cell number (23.1±2.2, 24.0±2.0, and 24.5±2.3 for the 0, 1, and 10 μM MG132 groups, respectively) on day 6 among the three groups. Interestingly, there was no difference in percentage of blastocysts between the 0 (18.5±4.7%) and 1 (25.1±4.7%) μM MG132 treatment groups; however, compared with the 10 μM MG132 group (14.0±4.7%), more embryos from the 1 μM MG132 group developed into blastocysts (p<0.05). To determine the effects on term development in vivo, two MG132 groups were included (0 and 1 μM MG132), and embryos were treated as above and transferred into synchronized surrogates after treatment. There was no difference in the oocyte–donor cell fusion rate, number of embryos transferred, pregnancy rate at days 28, 60, and at term, pigs delivered per embryo transfer, litter size, body weight at birth, nor cloning efficiency between the Scriptaid-alone control and MG132+Scriptaid combined groups. In summary, the combined treatment of MG132 and Scriptaid did not improve term development compared to Scriptaid treatment alone.
PMCID: PMC3459054  PMID: 22917492
17.  The In Vivo Developmental Potential of Porcine Skin-Derived Progenitors and Neural Stem Cells 
Stem Cells and Development  2012;21(14):2682-2688.
Multipotent skin-derived progenitors (SKPs) can be traced back to embryonic neural crest cells and are able to differentiate into both neural and mesodermal progeny in vitro. Neural stem cells (NSCs) are capable of self-renewing and can contribute to neuron and glia in the nervous system. Recently, we derived porcine SKPs and NSCs from the same enhanced green fluorescent protein (EGFP) transgenic fetuses and demonstrated that SKPs could contribute to neural and mesodermal lineages in vivo. However, it remains unclear whether porcine SKPs and NSCs can generate ectoderm and mesoderm lineages or other germ layers in vivo. Embryonic chimeras are a well-established tool for investigating cell lineage determination and cell potency through normal embryonic development. Thus, the purpose of this study was to investigate the in vivo developmental potential of porcine SKPs and fetal brain-derived NSCs by chimera production. Porcine SKPs, NSCs, and fibroblasts were injected into precompact in vitro fertilized embryos (IVF) and then transferred into corresponding surrogates 24 h postinjection. We found that porcine SKPs could incorporate into the early embryos and contribute to various somatic tissues of the 3 germ layers in postnatal chimera, and especially have an endodermal potency. However, this developmental potential is compromised when they differentiate into fibroblasts. In addition, porcine NSCs fail to incorporate into host embryos and contribute to chimeric piglets. Therefore, neural crest-derived SKPs may represent a more primitive state than their counterpart neural stem cells in terms of their contributions to multiple cell lineages.
PMCID: PMC3438846  PMID: 22482370
18.  T-Cell Immunophenotyping Distinguishes Active From Latent Tuberculosis 
The Journal of Infectious Diseases  2013;208(6):952-968.
Background. Changes in the phenotype and function of Mycobacterium tuberculosis (M. tuberculosis)-specific CD4+ and CD8+ T-cell subsets in response to stage of infection may allow discrimination between active tuberculosis and latent tuberculosis infection.
Methods. A prospective comparison of M. tuberculosis-specific cellular immunity in subjects with active tuberculosis and latent tuberculosis infection, with and without human immunodeficiency virus (HIV) coinfection. Polychromatic flow cytometry was used to measure CD4+ and CD8+ T-cell subset phenotype and secretion of interferon γ (IFN-γ), interleukin 2 (IL-2), and tumor necrosis factor α (TNF-α).
Results. Frequencies of CD4+ and CD8+ cells secreting IFN-γ-only, TNF-α-only and dual IFN-γ/TNF-α were greater in active tuberculosis vs latent tuberculosis infection. All M. tuberculosis-specific CD4+ subsets, with the exception of IL-2-only cells, switched from central to effector memory phenotype in active tuberculosis vs latent tuberculosis infection, accompanied by a reduction in IL-7 receptor α (CD127) expression. The frequency of PPD-specific CD4+ TNF-α-only-secreting T cells with an effector phenotype accurately distinguished active tuberculosis from latent tuberculosis infection with an area under the curve of 0.99, substantially more discriminatory than measurement of function alone.
Conclusions. Combined measurement of T-cell phenotype and function defines a highly discriminatory biomarker of tuberculosis disease activity. Unlocking the diagnostic and monitoring potential of this combined approach now requires validation in large-scale prospective studies.
PMCID: PMC3749005  PMID: 23966657
Mycobacterium tuberculosis; HIV; latent tuberculosis infection; active tuberculosis; biomarker
19.  Effect of Massage on the Efficacy of the Mental and Incisive Nerve Block 
Anesthesia Progress  2013;60(1):15-20.
The purpose of this trial was to assess the effect of soft tissue massage on the efficacy of the mental and incisive nerve block (MINB). Thirty-eight volunteers received MINB of 2.2 mL of 2% lidocaine with 1 : 80,000 epinephrine on 2 occasions. At one visit the soft tissue overlying the injection site was massaged for 60 seconds (active treatment). At the other visit the crowns of the mandibular premolar teeth were massaged (control treatment). Order of treatments was randomized. An electronic pulp tester was used to measure pulpal anesthesia in the ipsilateral mandibular first molar, a premolar, and lateral incisor teeth up to 45 minutes following the injection. The efficacy of pulp anesthesia was determined by 2 methods: (a) by quantifying the number of episodes with no response to maximal electronic pulp stimulation after each treatment, and (b) by quantifying the number of volunteers with no response to maximal pulp stimulation (80 reading) on 2 or more consecutive tests, termed anesthetic success. Data were analyzed by McNemar, Mann-Whitney, and paired-samples t tests. Anesthetic success was 52.6% for active and 42.1% for control treatment for lateral incisors, 89.5 and 86.8% respectively for premolars, and 50.0 and 42.1% respectively for first molars (P = .344, 1.0, and .508 respectively). There were no significant differences in the number of episodes of negative response to maximum pulp tester stimulation between active and control massage. A total of 131 episodes were recorded after both active and control massage in lateral incisors (McNemar test, P = 1.0), 329 (active) versus 316 (control) episodes in the premolars (McNemar test, P = .344), and 119 (active) versus 109 (control) episodes respectively for first molars (McNemar test, P = .444). Speed of anesthetic onset and discomfort did not differ between treatments. We concluded that soft tissue massage after MINB does not influence anesthetic efficacy.
PMCID: PMC3601725  PMID: 23506279
Dental pulp anesthesia; Lidocaine; Mental and incisive nerve block
20.  Cell Survival Signalling through PPARδ and Arachidonic Acid Metabolites in Neuroblastoma 
PLoS ONE  2013;8(7):e68859.
Retinoic acid (RA) has paradoxical effects on cancer cells: promoting cell death, differentiation and cell cycle arrest, or cell survival and proliferation. Arachidonic acid (AA) release occurs in response to RA treatment and, therefore, AA and its downstream metabolites may be involved in cell survival signalling. To test this, we inhibited phospholipase A2-mediated AA release, cyclooxygenases and lipoxygenases with small-molecule inhibitors to determine if this would sensitise cells to cell death after RA treatment. The data suggest that, in response to RA, phospholipase A2-mediated release of AA and subsequent metabolism by lipoxygenases is important for cell survival. Evidence from gene expression reporter assays and PPARδ knockdown suggests that lipoxygenase metabolites activate PPARδ. The involvement of PPARδ in cell survival is supported by results of experiments with the PPARδ inhibitor GSK0660 and siRNA-mediated knockdown. Quantitative reverse transcriptase PCR studies demonstrated that inhibition of 5-lipoxygenase after RA treatment resulted in a strong up-regulation of mRNA for PPARδ2, a putative inhibitory PPARδ isoform. Over-expression of PPARδ2 using a tetracycline-inducible system in neuroblastoma cells reduced proliferation and induced cell death. These data provide evidence linking lipoxygenases and PPARδ in a cell survival-signalling mechanism and suggest new drug-development targets for malignant and hyper-proliferative diseases.
PMCID: PMC3706415  PMID: 23874790
21.  Rapid Mobilization of Membrane Lipids in Wheat Leaf-Sheaths during Incompatible Interactions with Hessian Fly* 
Hessian fly (HF) is a biotrophic insect that interacts with wheat on a gene-for-gene basis. We profiled changes in membrane lipids in two isogenic wheat lines: a susceptible line and its backcrossed offspring containing the resistance gene H13. Our results revealed a 32 to 45% reduction in total concentrations of 129 lipid species in resistant plants during incompatible interactions within 24 h after HF attack. A smaller and delayed response was observed in susceptible plants during compatible interactions. Microarray and real-time PCR analyses of 168 lipid-metabolism related transcripts revealed that the abundance of many of these transcripts increased rapidly in resistant plants after HF attack, but did not change in susceptible plants. In association with the rapid mobilization of membrane lipids, the concentrations of some fatty acids and 12-oxo-phytodienoic acid (OPDA) increased specifically in resistant plants. Exogenous application of OPDA increased mortality of HF larvae significantly. Collectively, our data, along with previously published results, indicate that the lipids were mobilized through lipolysis, producing free fatty acids, which were likely further converted into oxylipins and other defense molecules. Our results suggest that rapid mobilization of membrane lipids constitutes an important step for wheat to defend against HF attack.
PMCID: PMC3586561  PMID: 22668001
22.  Mobilization of lipids and fortification of cell wall and cuticle are important in host defense against Hessian fly 
BMC Genomics  2013;14:423.
Wheat – Hessian fly interaction follows a typical gene-for-gene model. Hessian fly larvae die in wheat plants carrying an effective resistance gene, or thrive in susceptible plants that carry no effective resistance gene.
Gene sets affected by Hessian fly attack in resistant plants were found to be very different from those in susceptible plants. Differential expression of gene sets was associated with differential accumulation of intermediates in defense pathways. Our results indicated that resources were rapidly mobilized in resistant plants for defense, including extensive membrane remodeling and release of lipids, sugar catabolism, and amino acid transport and degradation. These resources were likely rapidly converted into defense molecules such as oxylipins; toxic proteins including cysteine proteases, inhibitors of digestive enzymes, and lectins; phenolics; and cell wall components. However, toxicity alone does not cause immediate lethality to Hessian fly larvae. Toxic defenses might slow down Hessian fly development and therefore give plants more time for other types of defense to become effective.
Our gene expression and metabolic profiling results suggested that remodeling and fortification of cell wall and cuticle by increased deposition of phenolics and enhanced cross-linking were likely to be crucial for insect mortality by depriving Hessian fly larvae of nutrients from host cells. The identification of a large number of genes that were differentially expressed at different time points during compatible and incompatible interactions also provided a foundation for further research on the molecular pathways that lead to wheat resistance and susceptibility to Hessian fly infestation.
PMCID: PMC3701548  PMID: 23800119
23.  Perfluorinated Compounds in Relation to Birth Weight in the Norwegian Mother and Child Cohort Study 
American Journal of Epidemiology  2012;175(12):1209-1216.
Perfluorooctane sulfonate and perfluorooctanoic acid are perfluorinated compounds (PFCs) widely distributed in the environment. Previous studies of PFCs and birth weight are equivocal. The authors examined this association in the Norwegian Mother and Child Cohort Study (MoBa), using data from 901 women enrolled from 2003 to 2004 and selected for a prior case-based study of PFCs and subfecundity. Maternal plasma samples were obtained around 17 weeks of gestation. Outcomes included birth weight z scores, preterm birth, small for gestational age, and large for gestational age. The adjusted birth weight z scores were slightly lower among infants born to mothers in the highest quartiles of PFCs compared with infants born to mothers in the lowest quartiles: for perfluorooctane sulfonate, β = −0.18 (95% confidence interval: −0.41, 0.05) and, for perfluorooctanoic acid, β = −0.21 (95% confidence interval: −0.45, 0.04). No clear evidence of an association with small for gestational age or large for gestational age was observed. Perfluorooctane sulfonate and perfluorooctanoic acid were each associated with decreased adjusted odds of preterm birth, although the cell counts were small. Whether some of the associations suggested by these findings may be due to a noncausal pharmacokinetic mechanism remains unclear.
PMCID: PMC3372312  PMID: 22517810
birth weight; MoBa; Norwegian Mother and Child Cohort Study; perfluorinated compounds; perfluorooctane sulfonate; perfluorooctanoic acid
24.  Novel genomic approaches unravel genetic architecture of complex traits in apple 
BMC Genomics  2013;14:393.
Understanding the genetic architecture of quantitative traits is important for developing genome-based crop improvement methods. Genome-wide association study (GWAS) is a powerful technique for mining novel functional variants. Using a family-based design involving 1,200 apple (Malus × domestica Borkh.) seedlings genotyped for an 8K SNP array, we report the first systematic evaluation of the relative contributions of different genomic regions to various traits related to eating quality and susceptibility to some physiological disorders. Single-SNP analyses models that accounted for population structure, or not, were compared with models fitting all markers simultaneously. The patterns of linkage disequilibrium (LD) were also investigated.
A high degree of LD even at longer distances between markers was observed, and the patterns of LD decay were similar across successive generations. Genomic regions were identified, some of which coincided with known candidate genes, with significant effects on various traits. Phenotypic variation explained by the loci identified through a whole-genome scan ranged from 3% to 25% across different traits, while fitting all markers simultaneously generally provided heritability estimates close to those from pedigree-based analysis. Results from ‘Q+K’ and ‘K’ models were very similar, suggesting that the SNP-based kinship matrix captures most of the underlying population structure. Correlations between allele substitution effects obtained from single-marker and all-marker analyses were about 0.90 for all traits. Use of SNP-derived realized relationships in linear mixed models provided a better goodness-of-fit than pedigree-based expected relationships. Genomic regions with probable pleiotropic effects were supported by the corresponding higher linkage group (LG) level estimated genetic correlations.
The accuracy of artificial selection in plants species can be increased by using more precise marker-derived estimates of realized coefficients of relationships. All-marker analyses that indirectly account for population- and pedigree structure will be a credible alternative to single-SNP analyses in GWAS. This study revealed large differences in the genetic architecture of apple fruit traits, and the marker-trait associations identified here will help develop genome-based breeding methods for apple cultivar development.
PMCID: PMC3686700  PMID: 23758946
GWAS; Linkage disequilibrium; Genetic architecture; Allele substitution effect; Pleiotropy; Malus × domestica
25.  Structural snapshots of the reaction coordinate for O-GlcNAc transferase 
Nature chemical biology  2012;8(12):966-968.
Visualization of the reaction coordinate undertaken by glycosyltransferases has remained elusive, but is critical for understanding this important class of enzyme. Using substrates and substrate mimics, we describe structural snapshots of all species along the kinetic pathway for human O-GlcNAc transferase, an intracellular enzyme that catalyzes installation of a dynamic post-translational modification. The structures reveal key features of the mechanism and show that substrate participation is important during catalysis.
PMCID: PMC3508357  PMID: 23103939

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