text:
StAR-related lipid transfer domain containing 7 (StarD7) is a member of the START-domain protein family whose function still remains unclear. Our data from an explorative microarray assay performed with mRNAs from StarD7 siRNA-transfected JEG-3 cells indicated that ABCG2 (ATP-binding cassette sub-family G member 2) was one of the most abundantly downregulated mRNAs.
Methodology/Principal Findings
Here, we have confirmed that knocking down StarD7 mRNA lead to a decrease in the xenobiotic/lipid transporter ABCG2 at both the mRNA and protein levels (−26.4% and −41%, p<0.05, at 48 h of culture, respectively). Also a concomitant reduction in phospholipid synthesis, bromodeoxyuridine (BrdU) uptake and 3H-thymidine incorporation was detected. Wound healing and transwell assays revealed that JEG-3 cell migration was significantly diminished (p<0.05). Conversely, biochemical differentiation markers such as human chorionic gonadotrophin β-subunit (βhCG) protein synthesis and secretion as well as βhCG and syncytin-1 mRNAs were increased approximately 2-fold. In addition, desmoplakin immunostaining suggested that there was a reduction of intercellular desmosomes between adjacent JEG-3 cells after knocking down StarD7.
Altogether these findings provide evidence for a role of StarD7 in cell physiology indicating that StarD7 modulates ABCG2 multidrug transporter level, cell migration, proliferation, and biochemical and morphological differentiation marker expression in a human trophoblast cell model.
Citation: Flores-Martín J, Rena V, Márquez S, Panzetta-Dutari GM, Genti-Raimondi S (2012) StarD7 Knockdown Modulates ABCG2 Expression, Cell Migration, Proliferation, and Differentiation of Human Choriocarcinoma JEG-3 Cells. PLoS ONE 7(8): e44152. doi:10.1371/journal.pone.0044152
Editor: Victor Sanchez-Margalet, Virgen Macarena University Hospital, Spain
Received: March 28, 2012; Accepted: July 30, 2012; Published: August 29, 2012
Copyright: © Flores-Martín et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was granted by the Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina (CONICET: PIP No 6352, PIP No 919), the Agencia Nacional de Promoción Ciencia y Técnica (FONCYT: PICT 2006/00147), the Ministerio de Ciencia y de la Provincia de Córdoba and the Secretaría de Ciencia y Tecnología de la Universidad Nacional de Córdoba (SECyT-UNC: 05/C244). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
StarD7 belongs to the family of START proteins ubiquitously expressed, which are implicated in lipid transport, metabolism, and signaling [1]–[3]. StarD7 mRNA was first identified using differential display techniques as a JEG-3 over-expressed gene compared with normal and benign trophoblastic samples [4]. In subsequent experiments, we demonstrated a predominant cytoplasmic localization of StarD7 in human cytotrophoblast cells with a clear and partial re-localization towards the plasma membrane after the syncytialization process [5]. Moreover, recombinant StarD7 accelerates bilayer fusion between donor and acceptor liposomes, induces multinuclear giant BeWo cell formation [6], and is able to form stable Gibbs and Langmuir monolayers at the air-buffer interface, showing a marked interaction with phospholipid monolayers [7]. A recent study reported that StarD7 protein facilitates the delivery of phosphatidylcholine to the mitochondria [8]. Also, we demonstrated that the regulation of StarD7 gene expression in JEG-3 cells occurs through a steroidogenic transcription factor 1 (SF-1) and β-catenin-mediated activation mechanism that synergistically activate StarD7 promoter [9], [10]. Subsequently, in order to get insight into the function of StarD7 in cell physiology, exploratory differential gene expression analysis of JEG-3 cells transfected with StarD7 siRNA was performed by microarray experiments. Data analysis indicated that ABCG2 was one of the most abundantly downregulated mRNAs [11].
ABCG2 is a member of the ABC protein superfamily of multidrug efflux transporters [12]. ABCG2 is an integral plasma membrane glycoprotein distributed in normal human tissues and particularly highly expressed in those with barrier function, including the placenta, testes, liver, kidney, intestine and brain [13], [14]. ABCG2 is overexpressed in tumors, cancer cell lines and in a subpopulation of stem cells: the side populations, conferring multidrug resistance [15], [16]. Alterations in ABCG2 expression linked with changes in cell proliferation, migration and invasion have been reported [17]–[19]. Besides the role as a drug and xenobiotic transporter or fetus protection against potential toxicity [20], [21], other physiological functions of ABCG2 in the placenta have been proposed [13], [14]. A role of ABCG2 in the transverse distribution of lipids in the plasma membrane during trophoblast syncytialization has been suggested [22]. In vitro trophoblast fusion and differentiation were accompanied with significantly increased in ABCG2 expression [23]; while inhibition of ABCG2 activity caused cytokine-induced trophoblast cell apoptosis [24]. Therefore, it has been suggested that the lower placental ABCG2 expression found in intrauterine growth retardation pregnancies may cause a deficit in placental function and survival [24].
In light of the above information, the present study was undertaken to establish the impact of StarD7 siRNA on ABCG2 expression in JEG-3 cells in connection with cell migration and proliferation. Moreover, phospholipids synthesis and biochemical and morphological JEG-3 cell differentiation markers were analyzed.
StarD7 siRNA Decreases ABCG2 mRNA and Protein Levels
To elucidate the impact of StarD7 siRNA on ABCG2 expression, JEG-3 cells were transfected with two different sets of double-stranded siRNA designed against different sequences of the StarD7 mRNA (Table 1). Among the two StarD7 siRNAs, StarD7.1 siRNA appeared a little more effective to inhibit StarD7 mRNA expression in JEG-3 cells, but no statistically significant difference among them was observed at all concentrations analyzed (p>0.05). After 48 h post transfection, approximately up to −79% and −60% (p<0.05) reduction of StarD7 mRNA expression was observed in JEG-3 cells treated with 200 nM of StarD7.1 and StarD7.2 siRNAs, respectively (Fig. 1A). In addition, approximately up to −83% and −63% (p<0.05) inhibition of StarD7 expression was detected by western blot with 200 nM of StarD7.1 siRNA or StarD7.2 siRNA, respectively (Fig. 1B). In the following experiments only the results obtained with StarD7.1 siRNA are shown, even though similar results were observed with StarD7.2 siRNA.
Cells were transfected with 100, 150 and 200 nM of StarD7.1 (siD7.1) or StarD7.2 (siD7.2) siRNAs and cultured for 48 h. Control cells were transfected with 200 nM of scrambled siRNA (siC). A-The StarD7 expression was determined by real-time quantitative PCR. Results are expressed as StarD7 mRNA expression in StarD7 siRNA-transfected cells after normalizing to cyclophilin A relative to the corresponding normalized mRNA levels in scrambled siRNA-transfected cells. The values represent the median and 25 th–75 th% percentiles of at least three independent experiments performed by triplicate. B- StarD7 protein expression was analyzed by western blot. Protein extracts (100 µg/lane) from scrambled siRNA- (siC), StarD7.1 siRNA- (siD7.1) or StarD7.2 siRNA- (siD7.2) transfected cells were electrophoresed on a 7.5% SDS polyacrylamide gel and transferred to a nitrocellulose filter. Filters were incubated with anti-StarD7Ct antibody (top) and with the monoclonal anti-α-tubulin antibody (bottom). A representative blot of at least five independent experiments with similar results is shown. The bar graph represents the densitometric quantification of StarD7 protein levels in StarD7 siRNA-transfected cells normalized to α-tubulin of five separate experiments relative to the corresponding normalized protein levels in scrambled siRNA-transfected cells defined as 1 (median and 25 th–75 th% percentiles). *p<0.05 compared to scrambled siRNA-transfected cells.
StarD7 knockdown led to a significant reduction in ABCG2 mRNA and protein levels in JEG-3 cells transfected with StarD7 siRNA as compared to scrambled siRNA-transfected ones. A significant inhibition of ABCG2 mRNA expression was observed in cells transfected with 200 nM of StarD7 siRNA cultured for 48 h or 72 h (Fig. 2A, p<0.05). In addition, western blot assays performed with protein extracts from StarD7 siRNA-treated cells revealed approximately up to −41% and −47% (p<0.05) diminution of ABCG2 protein level from 48 h of transfection, in parallel with a reduction in StarD7 protein level (Fig. 2C and 2D). Furthermore, immunofluorescence assays indicated that ABCG2 was localized to the plasma membrane of JEG-3 cells treated with scrambled siRNA but low or almost absent immunofluorescent ABCG2 signal was detected in the StarD7 siRNA-treated cells (Fig. 2B). Collectively, these data demonstrate that StarD7 siRNA results in a significant diminution of ABCG2 expression at both transcript and protein levels.
A- Cells were transfected with 200 nM of StarD7.1 or scrambled siRNAs and cultured for 48 h or 72 h. ABCG2 expression was determined by real-time quantitative PCR. Results are expressed as ABCG2 mRNA expression in StarD7 siRNA-transfected cells after normalizing to cyclophilin A relative to the corresponding normalized mRNA levels in scrambled siRNA-transfected cells. The values represent the median and 25 th–75th% percentiles of at least three independent experiments performed by triplicate. B- Confocal microscopy images demonstrating membrane expression of ABCG2 protein (red) in scrambled siRNA-treated JEG-3 cells (siC, upper panel) and low or absent ABCG2 immunostaining in StarD7 siRNA-treated cells (siD7, bottom panel). The nuclei were labelled with Hoescht (blue) and merge images are shown on the right. Bar = 20 µm (×600). C- Cells were transfected with 200 nM of StarD7.1 siRNA or 200 nM of scrambled siRNA and cultured until 96 h. ABCG2 protein expression was analyzed by western blot. Protein extracts (100 µg/lane) from scrambled siRNA- (siC) or StarD7 siRNA-transfected (siD7) cells were electrophoresed on a 7.5% SDS polyacrylamide gel and transferred to a nitrocellulose filter. Filters were incubated with anti-ABCG2 (top), anti-StarD7Ct (middle) and with the monoclonal anti-α-tubulin antibodies (bottom). These immunoblots are representative of at least three separate experiments. D- The bar graph represents the densitometric quantification of ABCG2 protein levels in StarD7 siRNA-transfected cells normalized to α-tubulin of at least four separate experiments relative to the corresponding normalized protein levels in scrambled siRNA-transfected cells (median and 25th–75th% percentiles). *p<0.05 compared to scrambled siRNA-transfected cells.
StarD7 siRNA Diminishes JEG-3 Cell Injury Repair, Migration, and Proliferation
Since it has been reported that alterations in ABCG2 expression are related with changes in cell migration and proliferation [17]–[19], we explored the effect of StarD7 reduction on cell migration performing monolayer wounding assay. Similar sized wounds were introduced in JEG-3 cell monolayers at 0 h. As shown in Figure 3A, in scrambled siRNA-treated cells the gap of the wound was gradually filled by migrating cells, and 24 h after wound the gap was almost closed. In contrast, transfection of StarD7 siRNA induced a decrease in cell migration. The speed of wound closure was much slower and the gap remained widely open after 24 h of culture. Figure 3B shows a quantitative analysis of pooled data, confirming that the remarkable difference in JEG-3 cell wound healing as a consequence of siRNA-mediated StarD7 knockdown was significant (p<0.05, n = 3). In addition, transwell assays with StarD7 siRNA-transfected cells showed a significant decrease in JEG-3 cell migration up to 40% of that observed in cells treated with scrambled siRNA (Fig. 3C). The quantitative analyses of pooled data document a clear inhibition of cell migration following StarD7 reduction.
A- Wound healing assay in JEG-3 cells treated with StarD7.1 (siD7) or scrambled (siC) siRNAs. An open furrow was generated by scratching confluent cells using a pipette tip. Confluency was restored in controls after 24 h. However, in cells treated with StarD7 siRNA, confluency was not restored after 24 h. B- The distance between furrow edges in the scrambled or StarD7 siRNA-treated cells of three independent experiments was measured and presented graphically as percentage of the initial distance (0 h);*p<0.05 compared to scrambled siRNA-transfected cells. C- Transwell In vitro migration assays. Left panels: representative images show cells migrated to the lower chamber after 48 hours (×200). Right panels: Bar graph represents the percentage of cell migration in seven fields of duplicate wells containing StarD7 siRNA-treated cells relative to scrambled siRNA ones (median and 25th–75th% percentiles, n = 3); *p<0.05 compared to scrambled siRNA-transfected cells. D-Cell proliferation was determined by BrdU (red) staining of JEG-3 cells treated with scrambled (left panel) or StarD7.1 (right panel) siRNAs. The nuclei were labelled with Hoescht (blue, middle panels) and merge images are shown on the bottom panels. Bar = 50 µm (×200). The images are representative of three experiments with consistent results.
To test whether this change in cell migration was accompanied with alterations in cell proliferation the BrdU uptake assay was performed. As shown in Figure 3D, there was a lower number of BrdU-positive cells in cultures treated with StarD7 siRNA than in scrambled siRNA-treated ones 40.7% (range 36.1–49.6%) vs 68% (range 64.1–74.0%), respectively (median and 25th–75th% percentiles, p<0.05, n = 3). Importantly, StarD7 reduction results in an inhibition of JEG-3 cell proliferation as indicated by the 3H-thymidine incorporation into StarD7 siRNA-treated cells compared to scrambled siRNA-treated ones. A significant reduction of −27% (25th–75th% percentiles: 18–41%, p<0.05) was obtained from three experiments performed in quintuplicate.
StarD7 Reduction is Associated with Decreased Phospholipid Synthesis
Mounting evidence indicates a strong connection among ABC transporter function and phospholipid synthesis, metabolism and signaling [25], [26]. Moreover, it has been reported that StarD2 promotes ATP-binding cassette protein A1-mediated efflux of cholesterol and phosphatidylcholine molecules as nascent pre-β-high-density lipoprotein particles [27]. Thus, to further determine the functional significance of the StarD7 reduction in JEG-3 cells we examined whether the de novo phospholipid synthesis is altered by StarD7 siRNA. For this aim, we examined 3H-glycerol incorporation into phospholipids in StarD7 siRNA- and scrambled-transfected cells after 48 hours of culture (Fig. 4A and B). Results show that there was a significant reduction in the biosynthesis of total phospholipids in JEG-3 cells treated with StarD7 siRNA compared to those treated with scrambled siRNA (Fig. 4A). However, the percentage of distribution of the major phospholipid species: phosphatidylcholine, phosphatidylserine, phosphatidylinositol, and phosphatidylethanolamine remained unchanged; being phosphatidylcholine the most abundant phospholipid synthesized in both experimental conditions (Fig. 4B). These results suggest that StarD7 downregulation leads to a global reduction in the synthesis of the main phospholipid species.
A- Relative 3H-glycerophospholipid synthesis in StarD7.1 (siD7) siRNA-treated JEG-3 cells compared to scrambled (siC) siRNA-treated cells defined as 1. Data are median and 25th–75th% percentiles (n = 4 independent experiments). *p<0.05 compared to scrambled siRNA-transfected cells. B- Percentage of distribution of major individual 3H-phospholipids in StarD7 siRNA-treated JEG-3 cells or in scrambled siRNA cells. Data are mean ± SEM (n = 2 independent experiments). Phosphatidylcholine (PC) is the species with the highest precursor incorporation in both cell conditions, followed by phosphatidylserine (PS) and after phosphatidylinositol (PI) and phosphatidylethanolamine (PE) with a minor percentage.
StarD7 siRNA Increases the Expression of Biochemical Differentiation Markers
It has been reported that ABCG2 silencing lead to a decrease in the expression of the biochemical differentiation markers syncytin and hCG in BeWo cells, protecting them during the period of transient membrane instability associated to biochemical differentiation and fusion events [22]. Thus, we next explored whether βhCG and syncytin-1 mRNAs levels are modified by knocking down StarD7. Surprisingly, quantitative RT-PCR analysis data showed an enhancement in both βhCG and syncytin-1 transcripts in StarD7 siRNA- compared to scrambled siRNA-transfected JEG-3 cells (Fig. 5, p<0.05). Next, we analyzed whether the increase in βhCG mRNA was also reflected in an increase in βhCG protein levels. A clear and significant increase in βhCG synthesis was observed in JEG-3 cells by StarD7 silencing at all time points analyzed (Fig. 6A and 6B, p<0.05). An evident negative correlation between StarD7 and βhCG levels was observed (Fig. 6A, middle panel). These results were also concurrent with an increase in βhCG secretion in StarD7 siRNA-transfected cells consistent with a biochemical differentiation of JEG-3 cells, compared to cells treated with scrambled siRNA (Fig. 6C, p<0.05).
Quantitative RT-PCR analysis of βhCG (A) and syncytin-1 (B) mRNAs in siRNAs-treated JEG-3 cells is shown. Analysis was performed using cDNAs derived from one µg of total RNA extracted from cells treated with StarD7.1 (siD7) or scrambled (siC) siRNA and cultured until 72 hours. Results are expressed as mRNA expression in StarD7siRNA-transfected cells after normalizing to cyclophilin A relative to the corresponding normalized mRNA levels in scrambled siRNA-transfected cells. The values represent the median and 25th–75th% percentiles of triplicate results obtained from at least three independent experiments; *p<0.05 compared to scrambled siRNA-transfected cells.
Cells were transfected with scrambled or StarD7.1 siRNAs for 6 h and then cultured until 72 hours. A- βhCG and StarD7 protein expression were analyzed by western blot. Protein extracts (100 µg/lane) from StarD7.1 siRNA-transfected (siD7) or scrambled siRNA-transfected (siC) cells were electrophoresed on a 7.5% SDS polyacrylamide gel and transferred to a nitrocellulose filter. Filters were incubated with anti-βhCG (top), anti-StarD7Ct (middle) and with the monoclonal anti-α-tubulin antibodies (bottom). These immunoblots are representative of at least three separate experiments. B- The bar graph represents the densitometric quantification of βhCG protein levels in StarD7 siRNA-transfected JEG-3 cells normalized to α-tubulin of five separate experiments relative to the corresponding normalized protein levels in scrambled siRNA-transfected cells defined as 1 (median and 25th–75th% percentiles). C- hCG secretion from JEG-3 cells after 48 h or 72 h of culture in StarD7.1 siRNA-transfected condition compared to scrambled siRNA-transfected cell culture defined as 1 (n = 5). *p<0.05 compared to scrambled siRNA-transfected cells.
StarD7 siRNA Increases Formation of Syncytial-like Structures
To explore whether the increase in βhCG and syncytin-1 expression was linked to alterations in cell morphology, desmoplakin staining was performed. As shown in Figure 7A StarD7 gene knockdown induced morphological changes in JEG-3 cells, promoting formation of syncytium-like structures. Even though a small cellular fusion index was detected in StarD7 siRNA-transfected cells (8%), it represents an almost three-fold increase as compared to cells transfected with scrambled siRNA (Fig. 7B, p<0.05).
Cells were transfected with scrambled (siC) or StarD7.1 (siD7) siRNAs for 6 h and then cultured until 72 hours. A- Detection of desmoplakin (red) in siRNA-treated JEG-3 cells was performed by immunofluorescence (left panel). The nuclei were labelled with Hoescht (blue) and merge images are shown on the right. Syncytial structures were characterized by the absence or incomplete desmoplakin staining (arrows). In control cells, desmoplakin labeling was continuous at the periphery of cells. Bar = 20 µm (×400). B- Percentage fusion was determined in cells transfected with StarD7.1 or scrambled siRNAs and represents the percentage of nuclei number in syncytia. Twenty fields were counted for each condition in three different experiments performed in duplicate as described in Materials and Methods. Results are depicted in terms of median percentage and 25th–75th% percentiles; *p<0.05 compared to scrambled siRNA-transfected cells.
StarD7 belongs to the START protein family widely expressed in human cell lines, with highest levels in JEG-3, JAR, HepG2, and HT-29 cell lines [4]. This family of proteins has been implicated in lipid transport, metabolism, and signaling [3], [28]. Human StarD7 orthologous genes have been annotated in the Ensembl and GenBank databases in different genomes, suggesting a conserved physiological function [29]. StarD7 protein has been predicted in most animal phyla, vertebrates and invertebrates as well as in plants highlighting its functional role. Even though some studies have been performed, its physiological function has not been fully elucidated [5], [8]. Here, we investigated the effect of reduced StarD7 expression by siRNA on different aspects of JEG-3 cell physiology. First, we established that StarD7 knockdown led to a decreased expression at both mRNA and protein levels for the ABCG2 xenobiotic/lipid transporter, concurrently with a significant diminution in cell migration, cell proliferation and phospholipid biosynthesis. Furthermore, βhCG mRNA expression as well as its protein synthesis and secretion were increased. This effect was correlated with a concomitant induction of the endogenous syncytin-1 mRNA level and a slight but significant reduction of intercellular desmosomes between adjacent JEG-3 cells. All together, these findings suggest that StarD7 modulates important aspects of trophoblast cell physiology.
The fact that ABCG2 expression was decreased after down-regulation of StarD7 draw our attention due to the important functions described for ABCG2 in cell physiology. The primary biological role of ABCG2 is to protect the organism from insults by a range of toxic, drug or carcinogenic xenobiotics, forming an important defense barrier. Apart from their function in drug transport and cell protection, ABCG2 and some other ABC proteins have been implicated to regulate phospholipid asymmetry by trafficking structural lipids within plasma membranes [25], [26], [30], [31]. Moreover, ABCG2 and ABCC1 participate in the secretion of the bioactive sphingosine 1-phosphate (S1P) [25]. Evseenko et al. demonstrated that the reduction of ABCG2 expression in BeWo cells by siRNA resulted in a manifest increase of phosphatidylserine externalization with ceramide accumulation. These effects were accompanied with a decrease of βhCG and syncytin mRNAs when cells were induced to differentiate by forskolin treatment; results that appear somehow in contradiction with the present data. Even though in both studies there was a down regulation of ABCG2 levels, it is important to emphasize that in the present report ABCG2 diminution was achieved after that StarD7 expression was knocked down. In addition, the observed discrepancies could be understood considering that Evseenko's study was performed on differentiating BeWo cells while undifferentiating JEG-3 cells were employed in the present study. Furthermore, both cell lines differ in several characteristics such as proliferative activity, degree of differentiation, cell motility, migration, and oxidative stress gene expression [32]–[34].
The specific mechanism by means StarD7 siRNA impact on JEG-3 cell migration and cell proliferation is unknown at present. However, it might be related to the reduction of ABCG2 expression. Indeed, several reports link alterations in ABCG2 expression with changes in cell migration, in addition to invasion and proliferation. Numerous studies documented that overexpression of ABCG2 correlates with higher migration and invasion in a variety of different tumor types [17], [35], [36]. On the contrary, inhibition of ABCG2 resulted in an impaired migration and tube formation of human microvascular endothelial cells [18] and also in inhibition of cellular proliferation in cancer cell lines [16]. Additionally, it was reported that hsa-miR-520 h downregulates ABCG2 in pancreatic cancer cells leading to inhibition of migration, invasion, and side population cells [19].
It has been established that ABCG2 activity is regulated by caveolin-1 through protein-protein interactions and also by membrane cholesterol micro-domain interactions [37], [38], underscoring the relevance of cellular lipid content, metabolism and distribution in ABCG2 function. Phospholipids are predominantly synthesized in the endoplasmic reticulum and subsequently transported to various destinations by vesicular transport through the fusion of vesicles to an acceptor compartment or can also be delivered to specific cellular organelles by monomeric exchange [39], [40]. In this regard, Horibata et al. reported that StarD7 mediates the delivery of phosphatidylcholine to mitochondria [8]. We have previously described that, in In vitro differentiating cytotrophoblast cells, StarD7 shows a partial re-localization towards the plasma membrane, suggesting that it could be implicated in the delivery of lipids to the cellular membrane [5]. Although we have not found modifications in the percentage of distribution of the main individual lipid species analyzed we cannot rule out a change in the amount of other minority compounds or alterations in the subcellular localization of particular phospholipids. Thus, it is possible to hypothesize that the observed phospholipid biosynthesis diminution is compatible with a compensatory mechanism aimed at reducing phospholipid accumulation, as a result of a decrease in phospholipid transport between organelles. Phosphatidylcholine, the main intracellular phospholipid, is metabolized to phosphatidic acid which in turn is converted to lysophosphatidate (LPA). Phosphatidic acid can also lead to sphingosine kinase-1 activation, which biotransforms sphingosine to S1P. LPA and S1P are survival signals that promote proliferation, migration, survival and angiogenesis [25]. In this regard, it has been reported a decline in the intracellular sphingosine concentration and sphingosine kinase 1 expression during trophoblast syncytialization [41], [42]. Moreover, the addition of S1P to cultured cytotropholasts led to a reduction in hCG secretion [42]. Our results indicate that the effect of StarD7 knockdown on total phospholipid biosynthesis diminution was accompanied with a decrease in cell migration and proliferation, and an increase in JEG-3 cell fusion and in the biochemical differentiation marker expression, βhCG and syncytin-1. In this scenario, even though we did not measure the intracellular level of S1P, it is possible to consider that phospholipid biosynthesis diminution led to a decline in S1P concentration which in turn stimulated the syncytialization process and also negatively regulated cell migration and proliferation. This hypothesis and our data are in line with the diminution in radiolabeled glycerol incorporation into the novo triacylglycerol and phospholipid biosynthesis during cytotrophoblast cell culture differentiation [43].
Herein, we observed that StarD7 downregulation in the choriocarcinoma JEG-3 cells induces cell fusion and expression of βhCG and syncytin. In contrast, in the context of normal cytotrophoblast cells that undergo spontaneous in vitro syncytialization, StarD7 mRNA and protein expression was increased [5]. These data suggest that an appropriate StarD7 level is required for normal cell physiology. There are several reports that support this assumption. First, StarD7 was originally identified as an up-regulated gene in the choriocarcinoma JEG-3 cell line with respect to their nonmalignant counterpart, complete hydatidiform mole and normal trophoblastic tissue [4]. Overexpressed StarD7 gene was one of the 147 genes specifically associated with colorectal tumor cells [44]. In addition, StarD7 upregulation has also been reported in B-chronic lymphocytic leukemia [45] and in multiple cancer cell lines [4]. Interestingly, Ikeda et al. found that miR-193b inhibits pancreatic cancer cell proliferation concomitantly with its ability to target and downregulate StarD7 transcript expression in pancreatic cells, effect associated with an inhibition in cellular proliferation [46]. Finally, StarD7 gene promoter is activated by Wnt/β-catenin signaling [10], a pathway that promotes proliferation and is frequently altered in cancer cells. These considerations and the observed induction of βhCG synthesis and secretion by StarD7 silencing are in line with the fact that several pathologic alterations in syncytiotrophoblast function have been associated to high hCG production and secretion [47]–[49]. Therefore, dysregulation of StarD7 expression could result in an altered trophoblast function or differentiation leading to an increased risk of placental disorders.
In summary, this study reveals that the loss of StarD7 protein in JEG-3 cells alters ABCG2 multidrug transporter level, cell migration, cell proliferation, and differentiation marker expression providing evidence for a new role for StarD7 in trophoblast cell physiology.
Materials and Methods
Mouse monoclonal anti-human ABCG2 (BXP-21 sc-58222) was obtained from Santa Cruz, polyclonal rabbit anti-human chorionic gonadotropin β-subunit (βhCG, A0231) from Dako. Anti-mouse and anti-rabbit IgG antibodies conjugated to horseradish peroxidase linked F(ab′)2 fragment (from sheep or from donkey respectively) were obtained from Amersham Bioscience. Mouse monoclonal anti-α-tubulin (Clone B-5-1-2), mouse anti-BrdU monoclonal antibody (B2531), and mouse anti-desmosomal protein (ZK-31) were obtained from Sigma Chemical Co. Anti-StarD7Ct was generated in our laboratory as described previously [5]. Two sets of double-stranded siRNA (designated StarD7.1 siRNA and StarD7.2 siRNA) were designed to target the position bases of the StarD7 coding sequence as indicated in Table 1 (Accession No. NM_020151). A scrambled siRNA sequence was used as a negative control.
Cell Culture and Knockdown of Endogenous StarD7
The human choriocarcinoma cell line JEG-3 (ATCC, HTB-36) was purchased from the American Type Culture Collection (ATCC, Rockville, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM), 10% (v/v) fetal bovine serum (FBS), 100 µg/ml penicillin, 100 µg/ml streptomycin (Invitrogen). The cells were harvested and seeded in 6 multiwell plates at 2×105 cells/well. After 24 h and at 40–50% confluency, cells were transfected (Lipofectamine 2000, Invitrogen) with 100 to 200 nM StarD7.1 or StarD7.2 siRNAs (Table 1), to knockdown endogenous StarD7 expression, or scrambled negative siRNA control (Silencer Negative™) (Applied Biosystems/Ambion, Biosystems, Argentina) in 0.5 ml of Opti-MEM serum-free medium. Following 6 h of transfection, 1.5 ml of DMEM was added and cells were cultured for 24–96 h. Culture medium was refreshed every 24 hours.
Quantitative Reverse Transcription-PCR
Total RNA was extracted from cultured cells using Trizol (Invitrogen), according to the manufacturer's instructions. Single-stranded cDNAs were synthesized with random primers (Invitrogen) in 20 µl of reaction. Briefly, one µg of total RNA was incubated with these primers (1.25 ng/µl), and the reverse transcriptase reaction was performed as previously described [4].
For real-time PCR, cDNA was mixed with 1× SYBR Green PCR Master Mix (Applied Biosystems) and the forward and reverse primers were added to a final volume of 15 µl. Primer sequences and concentrations used are listed in Table 2. Real-time PCR was carried out on an ABI 7500, Applied Biosystems with Sequence Detection Software v1.4. The cycling conditions included a hot start at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. Specificity was verified by melting curve analysis and agarose gel electrophoresis. Each sample was analyzed in triplicate. Transcript levels were normalized to those of cyclophilin A and relative expression levels were calculated using the 2–ΔΔCt method [50]. Amplification efficiency for each set of primers was near 98%. RNA samples incubated without reverse transcriptase during cDNA synthesis, as well as PCR reactions using water instead of template showed no amplification.
SDS-PAGE and Western Blotting
Protein samples were loaded onto a 10% SDS-PAGE gel. After migration, proteins were electrotransferred to nitrocellulose (Amersham Bioscience). The membrane was blocked in Tris buffered saline (25 mM Tris, 150 mM NaCl, 2 mM KCl, pH 7.4) containing 0.2% Tween 20 and 5% non-fat dry milk, washed and incubated with each one of the following primary antibodies: anti-StarD7Ct (0.5 µg/ml), mouse monoclonal anti-α-tubulin (1∶3000), rabbit polyclonal anti-βhCG (1∶1000), and mouse monoclonal anti-human ABCG2 (1∶300) for 1 h at room temperature with shaking, or as indicated by the manufactures. After washing, the blots were incubated with horseradish peroxidase-conjugated donkey anti-rabbit or sheep anti-mouse secondary antibodies (1∶5000) at room temperature for 1 h. Protein-antibody complexes were visualized by an enhanced chemiluminescence detection system (SuperSignal West Pico; Pierce). Blots were quantified by densitometry using Gel-Pro Analyzer. Protein expression was normalized to the α-tubulin expression.
βhCG Secretion
JEG-3 cells were treated with scrambled siRNA or StarD7 siRNA and cultured for 72 hours, with the media being replenished once at 24 hours after initial seeding. After 48 or 72 hours, the media was collected and the amount of secreted βhCG was quantified by a solid phase, two-site chemiluminescent immunometric assay with a detection limit of 1.0 mUI/ml (Siemens Immulite 2000).
Cell Migration Assay
JEG-3 cell migration was measured by determining the ability of the cells to move into an acellular space. The cells were treated with siRNA against StarD7 or scrambled siRNA as mentioned above. After 72 h of culture, the confluent monolayers were wounded using a sterile pipette tip and evaluated under phase contrast microscopy at 0, 8, and 24 h. Photographs were taken and the relative distance traveled by the cells at the acellular front was measured.
Alternatively, cell migration assay was carried out using the Transwell system (Cole Parmer) equipped with 8-mm pore size polycarbonate filters. Cells transfected with scrambled or StarD7 siRNA and cultured during 24 h were trypsinized, resuspended in their basal media containing 10% FBS and loaded onto the upper compartment of Transwell. The chambers were then placed into 24-well culture plates containing the basal media supplemented with 10% FBS. After 48 h of incubation, the non-migrating cells remaining in the upper compartment were removed using cotton swabs and the cells that had migrated to the lower surface of the filters were fixed with methanol and stained with Hoechst 33258. For each experiment, the number of cells in seven randomly chosen fields of each filter was counted. The results of three independent experiments are presented as percentage of cell migration relative to control.
Cell Proliferation Assay by Bromodeoxyuridine Uptake.
Proliferation was evaluated by analyzing the incorporation of BrdU into DNA. Cells were treated with siRNA against StarD7 or scrambled siRNA and cultured on coverslips as described above. After 72 h of culture, cells were exposed to 20 µM BrdU (Sigma) for 8 h, washed three times with phosphate-buffered saline (PBS), fixed 10 min in 3% paraformaldehyde, and washed three times with PBS. Cells were permeabilized for 10 min with 0.01% Triton X-100 in PBS (PBSTx). Cells were washed with PBS containing Tween 0.2% (PBST), treated with 2 N HCl at 37°C for 30 min, washed two times with 0.1 M NaB4O7, and then three times with PBSTx. Cells were resuspended in PBS containing 0.5% Tween 20 and 1% bovine serum albumin and incubated with mouse anti-BrdU monoclonal antibody at 1∶100 dilution for 1 h at 37°C. Then, cells were washed three times with PBST and incubated 1 h with Alexa Fluor 594-conjugated goat anti-mouse IgG (1∶1000). Cells were washed with PBST and slides were mounted in Aqueous Mounting Medium with fluorescence 361 tracers (Fluor Safe, Calbiochem).
Cell Proliferation by [3H]-thymidine Incorporation Assay
To measure the effect of StarD7 siRNA on cell proliferation, cells transfected either with scrambled or StarD7 siRNA were plated in quintuplicate at 1.5×104 cells/well in a 96-well plate, cultured during 72 h, and pulsed with [5′-3H] thymidine (20 µCi/ml, specific activity: 20 Ci/mmol) for the last 18 h of culture. Cells were harvested and washed with PBS. DNA was precipitated with cold 10% trichloroacetic acid (TCA), redissolved in 0.1 M NaOH with 1% SDS at 37°C for 1 h, and the radioactivity of the samples was counted in a Beckman liquid scintillation counter.
Immunofluorescence and Cell-fusion Assay
JEG-3 cells treated with siRNA against StarD7 or scrambled siRNA were cultured on coverslips as described above. Cells were fixed 10 min in cold methanol and incubated 10 min with 1 mM ammonium chloride to inhibit quenching. Cells were permeabilized for 7 min with PBSTx. Cells were then rinsed PBS three times, blocked with 2.5% normal goat serum in PBST and with 0.2% fish skin gelatin in PBSTx, and then incubated at 37°C with the mouse anti-desmosomal protein (1∶400) or mouse monoclonal anti-human ABCG2 (1∶100). Cells were washed with PBSTx and incubated 1 h with Alexa Fluor 594-conjugated goat anti-mouse IgG and nuclei were counterstained with Hoechst 33258 dye. Cells were washed with PBST and slides were mounted in Aqueous Mounting Medium with fluorescence 361 tracers (Fluor Safe, Calbiochem). Intercellular fusion was quantified by observing the coverslips using fluorescent microscopy, merging the Alexa Fluor 594 and Hoechst images, and counting the number of nuclei in syncytia and the total number of nuclei in twenty randomly chosen microscopic fields. The percentage of the nuclei in syncytia was determined as: (number of nuclei in syncytia/total number of nuclei) ×100, as described [51]. Duplicate wells were evaluated in each experiment, and each experiment was performed at least three times independently. Observations of cells were made in an inverted microscope Nikon Eclipse TE 2000U (Nikon Corporation, Japan) or a Confocal Olympus FLuoview FV300 microscope (Olympus Latin America, Miami, FL), as indicated.
Phospholipid Synthesis
The incorporation of 3H-glycerol into phospholipids in JEG-3 cells transfected with StarD7 siRNA or scrambled siRNA was assessed after 48 h of transfection. A 3 h labelling pulse of [2-3H]-glycerol (2 µCi/well; specific activity: 200 mCi/mmol) was given to cultures of each condition. The radioactive phospholipids were determined as described [52], [53]. In brief, cell preparations washed twice with 1 mM cold PBS were resuspended in 1 ml of water and precipitated with the same volume of 10% TCA and 1% phosphotungstic acid. After centrifugation (800×g, 15 min), the supernatant fraction was separated. Pellets were washed three times with 5% TCA and 0.5% phosphotungstic acid and once with water by successive resuspension and centrifugation. Phospholipids were extracted with chloroform/methanol (2∶1 vol/vol), and radioactivity was determined in a liquid scintillation counter. The content of protein in cell preparations was estimated by Bradford [54], using bovine serum albumin as standard. Individual phospholipids were separated by a 1-D two-solvent system procedure using high performance-TLC silica gel 60-precoated sheets with UV detector (Macherey-Nagel; Duren, Germany) as described by Weiss et al. [55]. Standards and labelled individual lipid species were visualized with iodine vapors, scraped from the silica-plate and the radioactivity in each lipid was quantified in a liquid scintillation counter.
Data Analysis
Significant differences for control and test conditions were identified using the nonparametric paired Wilcoxon test or unpaired Mann-Whitney U-test test. A Kruskal–Wallis with a Dunns post-test was performed to obtain a multiple comparison of independent sample populations. Significance was taken as p<0.05.
We gratefully acknowledge Dr. Cecilia Sanchez and Magalí Nazar for reagents and helpful advice. SG-R and GMP-D are Career Investigators of CONICET.
Author Contributions
Conceived and designed the experiments: SG-R GMP-D JFM. Performed the experiments: JFM VR SM. Analyzed the data: JFM VR SM GMP-D SG-R. Contributed reagents/materials/analysis tools: SG-R GMP-D. Wrote the paper: SG-R GMP-D.
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Very nice reflektion!BR!
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A fun grilled frank recipe with the frank spiral cut and served in a roll with ketchup and mustard.
CaloriesTotal FatProteinView Complete Nutrition Information >
Grilled Spiral Franks
A fun grilled frank recipe with the frank spiral cut and served in a roll with ketchup and mustard.
Recipe developed by George Duran
Prep Time(MINUTES)
Total Time(MINUTES)
3.9 of 5 (22 ratings) Review This Recipe | See All Reviews
- PAM® Grilling Spray
- 4 Hebrew National® Beef Franks
- 4 bamboo skewers, soaked
- 4 hot dog rolls, warmed
- 1/4 cup Hunt's® Tomato Ketchup
- 4 teaspoons Gulden's® Spicy Brown Mustard
Nutrition Information
Serving Size: 4 servings (1 sandwich each)
- Spray cold grate of gas grill with grilling spray. Preheat grill for medium-high heat. Meanwhile, carefully thread franks on skewers. To make spiral cut with paring knife, carefully cut down side of each frank while rotating skewer the entire length of the frank. Slightly pull frank apart to expose spiral shape.
- Grill 5 minutes or until franks are hot, turning occasionally. Remove franks from skewers. Place franks in rolls; add ketchup and mustard.
Cook's Tip
Soak bamboo skewers in water at least 30 minutes before using. Serve Alexia® Crinkle Cut Sweet Potato Fries with the grilled franks.
|Nutrition Information
|Amount per Serving
|% Daily Value*
|Total fat
|16 g
|Saturated fat
|6 g
|28 g
|Dietary fiber
|1 g
|7 g
|10 g
|Vitamin A
|Vitamin C
* Percent Daily Values are based on a 2,000 calorie diet.
About our Nutrition Information
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Ratings & Reviews
16 Reviews for Grilled Spiral Franks
I've never eaten a Hebrew National hotdog that needed anything more then yellow mustard,chopped onion and a good bun. BEST EVER !!!!
Jul 01, 2016
The recipe looks tastey and fun! All of the reviewers who commented on the skewer being "chewy" must either be joking or idiots. The instructions clearly state to remove the skewer. And, the spiral hot dog pictured on the bun has no skewer in it. Anyone with common sense would know to remove the skewer before eating the hot dog! I love Hebrew National Franks. They're the only franks I'll eat or feed to my family. And yes, there will be Hunt's ketchup available for those who enjoy it on their hot dog. We live in America where we have the right to put ketchup on our hot dogs if we so choose. Don't panic mustard fans, I always serve a variety of mustards with hot dogs as well: stoneground, spicy brown, Chipotle, classic French's - to name a few. If you choose to be a mustard purist at my house, you can be. If you like your dog loaded, I supply diced onion, relishes, and saurkraut too. It's all about your personal "pursuit of happiness". God bless America!
Jun 04, 2016
Instead of ketchup - use dill pickle relish and sliced onions - and mustard, of course. Yum!! Hebrew National are the best hot dogs. The very best.
May 26, 2016
Skewering the hot dogs to make a spiral dog is not explained properly. Insert a metal skewer into the center of the hot dog. Take a knife and make a spiral cut from top to bottom so the blade of the knife touches the skewer as you cut. Go back to the top of the hot dog and rotate it 1/3 of the way. Make another spiral cut trying to stay the same 1/3 distance away from the first cut. Repeat this step one more time. Remove the skewer and put the hotdog on the grill. Rotate it so that it gets a nice char on all those exposed edges as it cooks. This spiral cut will also completely expose the inside of the hot dog allowing the mustard or (Heaven forbid, ketchup) along with any toppings to get inside the dog instead of falling out of the sandwich bun. MANGIA!
May 25, 2016
Great idea. The grand kids loved it as did the adults. I used metal skewers. They slide out easier although they heat up on the grill.Use a light touch with the knife to avoid damaging the cutting edge. May dull the blade a little but that's why God gave us knife steels.
May 18, 2016
never have a hotdog good like that
Feb 10, 2016
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President Supports Minimum Wage Increase
The president urges a raise in minimum wage and reform of retirement savings plans.
The president urges a raise in minimum wage and reform of retirement savings plans.
BruceH: Holder is as misguided as most who think they can manage criminals or dictators. Holder had a pretty dismal record of letting the race of the criminal and wacky ideas like Fast and Furious get the best of him. Holder knows that the 1% ers where both misguided and in some cases criminals but he allowed them to deface our cities and parks. When Fast and Furious got out of hand, he seemed frozen. Then comes black lives matter and calls to kill white people and white police officers and the deaths of police through ambush double in one year. Who in this administration wants to take credit for this? And now he wants us to believe he suddenly changed his mind and wants the death penalty for police killers. Wow if you believe that I would suggest you get back on your meds.


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Cubs Negotiation List
Players selected in the MLB First-Year Player Draft (MLB Rule 4 Draft) are placed on a club's Negotiation List.
In most cases, a player selected by a club in the Rule 4 Draft will remain on the club's Negotiation List until either the player signs, or until 5:00 PM (EDT) on the Friday that falls during the week July 12-18 (whichever comes first), and if a club does not sign a Rule 4 Draft pick by the deadline, the player is removed from the club's Negotiation List and becomes eligible for selection again in the next Rule 4 Draft in which the player would be eligible for selection. The exception to this rule is any college senior with no baseball eligibility left. This class of player remains on the club's Negotiation List until the player signs or until one week prior to the next Rule 4 Draft (whichever comes first).
Also, any player eligible for selection in the Rule 4 Draft who is not drafted ("Non-Drafted Free-Agent" or "NDFA") can sign with any club at any time up until one week prior to the next Rule 4 Draft, unless the NDFA has college baseball eligibility remaining and is presently enrolled in a junior college or four-year college or enrolls in a junior college or four-year college subsequent to the Rule 4 Draft), in which case the July signing deadline applies.
A player selected in the MLB Rule 4 Draft or a Rule 4 eligible NDFA cannot be signed to a Major League contract.
A club is not permitted to select a player in the Rule 4 Draft two years in a row, unless the player gives his approval in advance.
CUBS NEGOTIATION LIST (updated 6-8-2013):
1. Kris Bryant, 3B (U. of San Diego) - JR
2. Rob Zastryzny, LHP (U. of Missouri) - JR
3. Jacob Hannemann, OF (BYU) - draft-eligible FR
4. Tyler Skulina, RHP (Kent State) - JR
5. Trey Masek, RHP (Texas Tech) - JR
6. Scott Frazier, RHP (Pepperdine) - JR
7. David Garner, RHP (Michigan State) - JR
8. Sam Wilson, LHP (Lamar CC) - JC3
9. Charcer Burks, OF (William B. Travis HS - Richmond, TX) - HS (NOTE: U. of Florida recruit)
10. Zack Godley, RHP (U. of Tennessee) - SR - NO COLLEGE ELIGIBILITY LEFT
11. Jordan Hankins, C (Austin Peay) - JR
12. Trevor Clifton, RHP (Heritage HS - Maryville, TN) - HS (NOTE: U. of Kentucky recruit)
13. Trevor Graham, RHP (Franklin Pierce) - JR
14. Daniel Poncedeleon, RHP (U. of Houston) - JR
15. Michael Wagner, RHP (U. of San Diego) - JR
16. Cael Brockmeyer, C (Cal State - Bakersfield) - JR
17. Kelvin Freeman, 1B (North Carolina A&T) - SR - NO COLLEGE ELIGIBILITY LEFT
18. Giuseppe Papaccio, SS (Seton Hall) - SR - NO COLLEGE ELIGIBILITY LEFT
19. Will Remillard, C (Coastal Carolina) - JR
20. Zak Blair, 2B (Mercyhurst College) - SR-5th - NO COLLEGE ELIGIBILITY LEFT
21. Josh McCauley, RHP (Shepherd U.) - SR - NO COLLEGE ELIGIBILITY LEFT
22. Kevin Brown, LF (Bryant U.) - SR - NO COLLEGE ELIGIBILITY LEFT
23. Tyler Ihrig, LHP (Marin CC) - JC2
24. Tyler Alamo, C (Cypress HS - Cypress, CA) - HS (NOTE: Cal State - Fullerton recruit)
25. Marcus Doi, OF (Mid-Pacific Institute - Honolulu, HI) - HS (NOTE: U. of Hawaii recruit)
26. Carlos Pena, C (Southwest Miami HS - Miami, FL) - HS
27. Tyler Sciacca, 2B (Villanova) - SR - NO COLLEGE ELIGIBILITY LEFT
28. Brad Renner, RHP (Florida State College) - JC2
29. John Garcia, CF (Denbigh HS - Newport News, VA) - HS
30. Zak Hermans, RHP (Princeton) - SR - NO COLLEGE ELIGIBILITY LEFT
31. Sean Johnson, RHP (Iowa Western CC) - JC1
32. Keaton Leach, RHP (Glendale CC) - JC1 (NOTE: Fresno State recruit)
33. Chris Madera, CF (Northwest Florida State College) - JC2
34. Jake Thompson, RHP (Siuslaw HS - Florence, OR) - HS (NOTE: Oregon State recruiit)
35. Ramsey Romano, SS (Valhalla HS - El Cajon, CA) - HS (NOTE: U. of Michigan recruit)
36. Derek Campbell, SS (U. of California) - JR
37. Jeremy Martinez, C (Mater Dei HS - Santa Ana, CA) - HS (NOTE: USC recruit)
38. Zack Brown, RHP (Seymour HS - Seymour, IN) - HS (NOTE: U.of Kentucky recruit)
39. Josh Greene, CF (Forest HS - Ocala, FL) - HS (NOTE: High Point U. recruit)
40. Bubby Riley, LF (Delgado CC) - JC2 (NOTE: NC State recruit)
fwiw, it's very unlikely b
fwiw, it's very unlikely b.riley(40th) doesn't choose to play for NCSU.
strong kid, btw. you can tell he doesn't skip the gym too much.
he's got a bit of speed and good arm (can easily play RF) to go with his decent (though not mind-blowing) power.
he turns 21 soon...he should raise his draft value easily next season. if the cubs snag him it's a sneaky nice pick that late in the draft.
Cubs sign 12th round pick,
Cubs sign 12th round pick, Trevor Clifton, Highschool RHP, Knoxville TN; 6'5" 185#. Supposedly a tough sign because of his commitment to Univ. of Kentucky...hence the "3rd round money".
"It was a dream come true," Clifton said about being drafted. "I got the money I wanted, and playing professional is a dream."
Clifton didn't disclose his asking price but said he asked for third-round money. "And that's what I got," he said.
Clifton will report to AZ Phil in Mesa (in a week).
Clifton will report to AZ
Clifton will report to AZ Phil
I hope he won't be expected to have the same attention to detail. If so, he may as well stay home.
a bit dated but I read this
a bit dated but I read this brief note on pg.5 of the Baseball America magazine from May 28th-June 11th (Draft Preview issue with Clint Frazier on the cover):
Paniagua Locked Out
One month into the minor league season, Cubs righthander Juan Carlos Paniagua had still not received his visa to travel to the US.
Paniagua signed out of the Dominican Republic for $1.5M last year in July, hd his contract approved and even pitched in teh US breifly after signing. However, the US Consulate has been requesting documents from Paniagua--including school records and identifying documents from his siblings--before issuing him a visa. There's no clear time-table for when Paniagua might arrive in the Us to begin his first full minor league season. Paniagua has a checkered history with MLB investigations, having failed two of them previously and served two separate penalties that banned him from signing for one year.
I think I read some comment that he might play in the Dominican league while waiting for his Visa. Anyone hear anything else?
Huh, until now, I was never
Huh, until now, I was never aware that an undrafted junior at a 4-year college could sign as an NDFA once his junior season is over.
if you're 21 or over, you're
if you're 21 or over, you're free game...which is how they drafted that college freshman at BYU.
I believe you're actually
I believe you're actually eligible for the draft if you turn 21 within 45 days of the draft. I was just unaware that that rule applied to NDFA too.
Good to know - now I'll fret about a couple of juniors who weren't drafted from my school.
ah, gotcha.
ah, gotcha.
I believe it applies to High
I believe it applies to High School kids who just graduated too but have yet to attend classes in college. (Think Jesse Hodges).
But they would be subject to the 11th to 40th round bonus rules. So $100K or whatever is it and anything over that comes out of your top 10 picks allotment.
(AZ Phil, please correct me if I'm wrong.)
Hodges story: http://mlb.mlb.com/news/article.jsp?ymd=20121219&c...
UCLA similarly lost a high
UCLA similarly lost a high profile recruit - Felipe Perez - to the Diamondbacks last year. He was so unlikely to sign no one drafted him but after he did well in a summer league, the DBacks, who apparently had bonus space left, signed him for $400,000.
e.jackson throwing his best
e.jackson throwing his best game since his 1st game as a cub...and still losing.
6ip 4h 1bb 6k, 1r/er - 76 pitches
hitting 96mph on the radar at the high end of his velocity...more often 94mph.
7ip 4h 1bb 8k - 1/er...and a
7ip 4h 1bb 8k - 1/er...and a win.
rizzo cracks his bat...and
rizzo cracks his bat...and unlike almost all of MLB...though it's suggested/advised by MLB...he has a replacement waiting for him in the on-deck circle.
...and cody ransom goes 1st to 3rd on a groundout to 1st thanks to the shift on rizzo leaving no one covering 3rd. lulz.
tie game on a sac fly!
tie game on a sac fly!
...screw you shift.
...and the no-hitter broken up after 5.2ip
ransom 3r HR! 1-4, bottom 7.
ransom 3r HR!
1-4, bottom 7.
anyone know what's with Mike
anyone know what's with Mike Olt? He's hitting .162 (according to the box score vs I-Cubs today). About 110 at bats...he catch Ian Stewart disease?
The Round Rock lineup sported Robinsin Chirinos leading off and Jim Adduci batting 2nd.
Junior Lake continued his hot hitting and went 3-5 including a double, 2 runs and 1 rbi.
Daytona won 8-2 with Baez and Solar hitting doubles, Geiger with 2 hits and a HR.
Patrick Kane Co. Cougars lost but Vogelbomb had a double and a HR and Almora had two more hits.
Olt was having some vision
Olt was having some vision problems and spent time on the DL. They activated him about a week ago so maybe they've figured out a fix.
he's been experiencing
he's been experiencing "vision issues" since spring...at one point going on the DL for it.
when he returned from the DL nothing improved, though.
Lake passes the eyeball test
Lake passes the eyeball test w/ flying colors...made some nice plays the other night in RF but has already racked up 5 errors @ 3B...gonna drive over to cedar rapids tomorrow for a matinee look @ AA & Co. before he moves up...
The Cubs also have Jae-Hoon
The Cubs also have Jae-Hoon Ha as an all-outfield right-handed hitter who I project to be eventually good enough for an MLB job. Presumably, the Cubs would only carry one of him or Lake.
I hadn't really considered the effect that Soler and Almora would have on the roster makeup, but presumably there would be at most one roster slot for a guy like Lake or Ha.
1. Go Blackhawks
1. Go Blackhawks
2. I don't get the Bears trading Carimi for a 6th round pick, well I get it, better to get something than nothing, but seems like it would be worth seeing if he's recovered from the knee injury.
3. oh yeah Cubs, wtf is up with Castro?
i wish they'd give castro a
i wish they'd give castro a day off rather than pretending he's going to be the next cal ripken jr.
dunno how much it would help...but he's playing really tired baseball. he's had 1 hit this month...
i've never expected much out of him except to be a .280/.330 avg/ob% type hitter with 30+ doubles, a handful of triples, and 15+ HR (which isn't bad for a SS).
still...he's not even scraping that since april, though he did hit a decent amount of doubles in may...
I'd like to see them sit him
I'd like to see them sit him for a few days and have him do nothing those days except take batting practice with the hitting coach and take some infield practice. Time to reboot.
well he sounds hopeless...
well he sounds hopeless...
"Every time I'm going to be aggressive," Castro said. "To go up there and take a walk – not everybody has that ability. I want to go up there and swing. And when I look at the video, (I'm doing) the same thing I did before (when I hit .300). I'm just a little bit slow. I keep working. I know I'll get out of it, especially now, maybe hitting seventh (will help). If (this goes) good, then it's back to second and stay hot again."
we're talking about a guy who
we're talking about a guy who struggles to walk 35 times a season. he didn't walk in the minors...he's not going to start now.
the ability to take a walk (or not) never has been part of his game and probably never will be...quality contact and bit of luck hitting the ball where the defenders aren't is the type of hitter he is. if anyone was expecting him to progress in this part of his game based on his age, i dunno what to say. that's not his approach...he thinks he can square up anything...he doesn't have that "wait for my pitch" plate approach. i doubt seriously he could learn how given the stuff he swings at...both what goes for a hit and what gets hit into an out.
I agree completely. He models
I agree completely. He models himself after Vlad Guerrero, which is fine if you hit like Vlad did for awhile. But as soon as you lose bat speed, which Castro doesn't seem to have done, or your mechanics are off, which they seem to be, you're hosed. Randall Simon had an exaggerated version of the Castro approach and had a nice year with the Cubs. Man, that guy swung at pitches even more out of the zone than Castro and clubbed some shots. It was almost funny.
Sveum mentioned at some point
Sveum mentioned at some point that Castro has an extra leg kick he wants to eliminate so he can sit back more, so it seems like there's a plan, but I doubt anything major like that would happen during the season.
Castro on Castro: And when I
Castro on Castro: And when I look at the video, (I'm doing) the same thing I did before (when I hit .300).
Sveum on Castro:
I'd like to see him back to where he was in 2010 and 2011 when there was a lot less movement going on. He thinks it's the same mechanics, but they're not the same mechanics from when he came up. It might look the same but there's way more movement going on."
2. I didn't get that at all.
2. I didn't get that at all. That seems like a trade that says, "this guy is no good." I have to assume that the new coaching staff thinks he's, well, no good.
Teams don't take kindly to
Teams don't take kindly to players skipping the "optional" team activities (OTAs). Plus they think he's no good. So instead of getting nothing for him by releasing him, they got a draft pick.
This is actually a decent
This is actually a decent article from ESPN about Edwin Jackson
Jackson's .358 BABIP puts him on pace to set a new career high and is nearly fifty points higher than his career average of .309.
At least he had a decent game
At least he had a decent game last night. I was beginning to think that the next thing we were about to hear was, "Jackson visited Dr. Andrews today and was told..."
I just did the math and found
I just did the math and found that if his BABIP had been .300 this season, assuming no other changes, his WHIP would be 1.35 instead of 1.57. Sequencing would still be a key factor in determining his ERA, but simply on a scaled measure, his ERA would drop from 5.76 to 4.95. All this says to me that he has been unlucky, yes, but also not as effective as a $13 million pitcher should be despite that.
Jim [email protected]
He's not called QuietMan for
He's not called QuietMan for nothing.
not a Cub within site of the All-Star voting
I presume the only 2 options are Rizzo (Goldschmidt and Votto should be locks) and Feldman...maybe Gregg.
ninja/wood/feldman/gregg are
ninja/wood/feldman/gregg are the only legit candidates right now...whatever the manager seems to want to fit into his roster.
outside shot of j.russell or rizzo...but that would be a surprise.
If I'm managing, I take Ninja
If I'm managing, I take Ninja, save him to pitch in extra innings or to pinch run.
ugh, totally blanked on
ugh, totally blanked on Travis Wood, slightly better numbers all around than Feldman at the moment. 3-7 Ninja is going to be tough to justify with 2 guys on his own team with better WHIP's and ERA's and records.
Cub picks 11-40
Jim Callis: A for Cubs.
Jim Callis: A for Cubs.
FWIW Callis on 670 The Score really liked the Cubs draft including the pitchers right behind Bryant whom he deemed great value picks.
Gave the Sox a B-
He said he recommended Gabe Carimi to Jerry Angelo, too.
I made the last one up.
Recent comments
Torres a late scratch from the Pelicans game and no word of injury or signs of foul play with the unis. May be a trade a brewing after all?
Edit: now word he was a late scratch due to a "headache". Hmmm
Hopefully Zobrist is regaining his stroke and KB can move back down to third or fourth in the order. Did not realize the zero rbi's in over a week - glad Rizzo and Russell picking up the slack
Just had to ask, I guess.
Haven't had a big comeback win in a long time (at least it seems like a long time). Great feeling!
Rizzo should hold clinics on 2-strike hitting. Awesome.
cubs lead in the top 7th!
so nastay...
triple, walk, domination
And he gets out of it by K-ing Braun, Lucroy and Carter!
Let's get some runs!
welcome to the cubs j.nathan.
2 pitches into the inning and there's a guy on 3rd with 0 outs (triple).
his velocity is hanging around 91/92mph.
Always a good idea to bring in Richard against a LH batter.
Russell left game with left heel contusion from foul ball on Saturday. Started to bother him during the Sunday game.
a.russell out and down into the clubhouse...no one knows why.
it's very hot on the field, so it might be heat related.
I blame the All-Star game again, or else Michael Barrett.
Miller park roof is closing. Hmmm, might play differently now?
Lester at 72 pitches through 3. Roof closing might get him through 6.
Middlebrows pulls a calf or hamstring. Len says he blew a tire rounding 1st. Nieuwenhuis enters game.
Now roof opening. Dizziness. No AC in the ballpark wrt the field.
just to lighten the mood:
Shark's day: 5.2 IP 5 Runs (all earned), 8 Hits, 2 HR. Left the game down 5-0 to Yankees.
Anyone expecting game 4 NLCS Shark vs Hammel or Lackey?
First inning: two on, no out, Rizz and Zobrist due up. Don't score.
Third inning: two on, one out, KB and Rizz due up. Don't score.
This is getting' old, fellahs.
KB still with ZERO RBI since the A/S break. Ouch.
62 through 2ip...yow.
it's an off day for d.ross, too. the wild pitch + passed ball in 2 separate strike 3 calls allowing the runner to reach 1st and the 5sb through 2ip makes for a bad game so far. ross cant shoulder any blame for some of those SBs, especially that villar steal of 3rd while lester watched him.


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Abbotsford Information and Schedule
Abbotsford tickets are available now for all Abbotsford events. Whether on vacation or a current resident, you'll never get tired of all the things that are available in Abbotsford, British Columbia, and the surrounding areas! Get tickets to Abbotsford, BC below.
All Events by Date
|Event & Venue/City
|Aug 27, 2016
Sat 8:00PM
|Punjabi Virsa
|Sep 9, 2016
Fri 6:45PM
|Five Finger Death Punch, Papa Roach & Sixx: A.M.
|Sep 18, 2016
Sun 7:00PM
|Blink 182, A Day To Remember & All American Rejects
|Oct 15, 2016
Sat 7:00PM
|Our Lady Peace & I Mother Earth
|Oct 18, 2016
Tue 7:30PM
|John Fogerty
|Oct 20, 2016
Thu 7:00PM
|Slayer, Anthrax & Death Angel
|Nov 16, 2016
Wed 7:30PM
|Mamma Mia!
|Nov 23, 2016
Wed 7:30PM
|Celtic Thunder
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Presbyterian Women is a national organization within the Presbyterian Church (U.S.A.). The group works to support women spiritually through Bible study as well as providing a place for fellowship and opportunities for education and mission. The women of Cottonwood gather regularly and invite you to participate with them.
Prayer Shawl Ministry - Do you know how to knit and/or crochet? The Prayer Shawl Ministry needs your help to knit or crochet prayer shawls for CPC and hospice use and blanket squares for the Uganda Fistula Hospital surgical patients. Yarn and instructions are available for you to use. The Prayer Shawl group of Presbyterian Women meets the first Wednesday of each month from 1:00-2:30 p.m. in room one of CPC. Donations of crochet hooks, 10 inch knitting needles sizes 6-10 and new machine washable yarn are also welcomed, so clean out those craft closets, boxes and drawers, and bring your unwanted yarn, needles and hooks to CPC. Questions? Ask Barbara White.
Call Sue Martin, 801-209-3062 as to day, meeting time and place
Your Cottonwood PW wish to notify all women of Cottonwood Presbyterian Church that we are members of P.W. Worldwide, an organization that encompasses the world and yet so close on Vine St. You are needed and wanted at all our functions. Looking forward to a new year of gatherings to serve our church. With your help we will have a successful, wonderful year of fellowship, mission work and growth.
A suggestion box will be on the P.W. board for suggestions on how we can meet your needs and program ideas.


text:
LifeNet of New York
We are a community based program that provides air medical transport service throughout New York, Pennsylvania, Vermont, Massachusetts and Connecticut. We have ten aircraft strategically located across our service area.
Want updates on LifeNet of New York? Join our mailing list!
2016 CAMTS Public Notice - Click Here.


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Amazon has this KidKraft Vintage Play Kitchen in Blue for just $81 shipped right now — the lowest price on record, by far!
Sign up for a free trial of Amazon Prime to get free 2-day shipping.
Psst! Don't forget you can sign up for Swagbucks to earn free gift cards to use on deals on Amazon.
Thanks, Passionate Penny Pincher!


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16.06.2016 18:11:00
Belarus test fires long-range MLRS Polonez
The first live test of the multiple-launch rocket system (MLRS) Polonez has taken place in Belarus. The first live fire test of the multiple-launch rocket system Polonez took place in Gomel Oblast on 16 June. As part of the life fire test unmanned aerial vehicles were used to feed real-time data about the target to the multiple-launch rocket system.
Full text
Goscomvoyenprom Collegium Discusses Implementation of Business Plans for Development of Companies25.04.2016 16:38:00
On 22 April 2016, AGAT-Control Systems, management company of the Geoinformation Control Systems Holding hosted a meeting of the collegium headed by Chairman of Goscomvoyenprom Sergei Gurulev. The meeting was attended by heads and directors of Goscomvoyenprom companies and departments, their deputies for economics (chief economists) and chief engineers.
Goscomvoyenprom Chairman Meets with Indonesian Defence Minister29.04.2016 16:41:00
A meeting between Goscomvoyenprom officials and the Indonesian delegation took place on 28 April. The parties confirmed their mutual interest in the development of bilateral military-technical cooperation, discussed the implementation of agreements reached earlier, noted the potential and identified specific targets for the expansion of cooperation in a number of areas in the near future.
Visit to ground control station of Belarus' Satellite Communication and Broadcasting System12.04.2016 15:27:00
Belarus intends to follow the way of getting integrated into the global information space by developing the national satellite communication and broadcasting system. Belarus President Alexander Lukashenko gave the relevant instructions as he visited the space communication center in the town of Stankovo, Dzerzhinsk District, Minsk Oblast, on 12 April.
Radionavigation Interstate Council Considers Interstate Radio-Navigation ministries. Programme Till 2016 and Adopts the Draft of New 2020 Programme09.06.2016 10:50:00
On 8–9 June 2016, AGAT – Control Systems and NTLab-IS, Minsk (Belarus), hosted a regular meeting of national state customers of the CIS Interstate Radio-Navigation Programme between the CIS member states to 2016 (IRNP 2016), the 46th meeting of Radio-Navigation Interstate Council and meeting of the Radio-Navigation sci-tech council on selecting tasks to the new interstate programme to 2020. The events were attended by representatives were analysing implementation of IRNP 2016 and synchronised eff orts on successful completion of the planned undertakings.
Mikhail Demchenko Was Elected Director of AGAT-Control Systems15.06.2016
On 13 June, the Belarusian President Alexander Lukashenko gave his consent to appoint Mikhail Demchenko Director of AGAT-Control Systems, who from August 2014 had held the position of Deputy Director for Commercial Matters.
On 13 June, by the decision of the Supervisory Board of Directors of AGAT-Control Systems Mikhail Demchenko was elected Director of the company. From the 14th of June he assumed his duties.
Belarusian Volat presents armored truck at Eurosatory 201614.06.2016
The Belarusian automobile engineering company Volat (OAO Minsk Wheel Tractor Plant or MZKT for short) presented a breadboard model of the lightly armored automobile V-1 during the international defense and security expo Eurosatory 2016 in Villepinte, France.
Gennady Smolsky Was Appointed Deputy Chairman of Goscomvoyenprom14.06.2016
Gennady Smolsky Was Appointed Deputy Chairman of Goscomvoyenprom President of Belarus Alexander Lukashenko signed a relevant decree on 13 June.


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|Brand Name
|Bel-Art Products
|Item Weight
|4.8 ounces
|Product Dimensions
|6.5 x 4.7 x 2.6 inches
|Item model number
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|Manufacturer Part Number
This polypropylene system is strong enough to support the weight of even the heaviest pipettor. Mix and match components to suit your needs. Single holder clamp, package of 2. Attach to any shelf or benchtop edge up to 2.54cm (1") thick using the large locking thumbscrew clamp. Supports many standard brands of pipettors. All components are chemical resistant and autoclavable at 121ºC (250ºF).


text:
Original Article
The Limitations of Pulsed-Field Gel Electrophoresis for Analysis of Yersinia enterocolitica Isolates
Version of Record online: 15 NOV 2013
DOI: 10.1111/zph.12085
© 2013 Blackwell Verlag GmbH
Zoonoses and Public Health
Volume 61, Issue 6, pages 405–410, September 2014
Additional Information
How to Cite
Gilpin, B. J., Robson, B., Lin, S., Hudson, J. A., Weaver, L., Dufour, M. and Strydom, H. (2014), The Limitations of Pulsed-Field Gel Electrophoresis for Analysis of Yersinia enterocolitica Isolates. Zoonoses and Public Health, 61: 405–410. doi: 10.1111/zph.12085
Publication History
- Issue online: 16 JUL 2014
- Version of Record online: 15 NOV 2013
- Manuscript Received: 11 MAR 2013
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text: Energy may be stored for use during peak demand by pumpingwater to a high reservoir when demand is low and then releasing itto drive turbines when needed. Suppose water is pumped to a lake135 m above the turbines at a rate of 1.00 x 105 kg/sfor 10.0 h at night. (a) How much energy (kWh) is needed to do thiseach night? (b) If all this energy is released during a 14-h day,at 75% efficiency, what is the average power output?