LINE-1 Based Insertional Mutagenesis Screens to Identify Genes Involved in Embryonic Stem Cell Differentiation

Background: Knowledge of the intrinsic properties of embryonic stem cells is essential before the possibility of using these cells for therapeutic purposes becomes a reality. Insertional mutagenesis screens are widely used to identify genes sufficient to confer a particular phenotype; however, applications of such approaches are limited to mouse models. Thus, there is a need to develop a new DNA transposon system that allows gene discovery approaches in stem cells. Methods: This study was performed to explore the possibility of using the long interspersed nuclear element 1 (LINE -1) retrotransposon as a gene trap vector to identify genes involved in the maintenance and differentiation of mouse embryonic stem cells. Results: We developed an episomal, nonviral LINE-1 retrotransposon system using the scaffold/matrix attachment regions in the backbone of our vector. This gene trap vector harbors a GFP marker whose expression occurs only after successful insertional mutagenesis. By utilizing this vector, coupled with GFP expression, we have successfully isolated four individual embryonic stem cell clones that display disrupted genes, including two known genes. We then confirmed the identity of these genes using an inverse PCR approach and verified their function in cell differentiation using shRNAs and undifferentiated markers of embryonic stem cells. Conclusions: The ease of using this insertional mutagen and the simplicity of identifying the cells with disrupted genes by GFP expression make this LINE-1 vector a promising tool for embryonic stem cell and cancer stem cell gene discovery

Towards a Typology of Learning Workers: Latent Class Analysis of Adult Education Participation in the United States

This study identifies a typology of learning workers by investigating profiles of individuals participating in adult education. Using latent class analysis, we examined four classes and their associations with covariates

The Singer's Formant and Speaker's Ring Resonance: A Long-Term Average Spectrum Analysis

ObjectivesWe previously showed that a trained tenor's voice has the conventional singer's formant at the region of 3 kHz and another energy peak at 8-9 kHz. Singers in other operatic voice ranges are assumed to have the same peak in their singing and speaking voice. However, to date, no specific measurement of this has been made.MethodsTenors, baritones, sopranos and mezzo sopranos were chosen to participate in this study of the singer's formant and the speaker's ring resonance. Untrained males (n=15) and females (n=15) were included in the control group. Each subject was asked to produce successive /a/ vowel sounds in their singing and speaking voice. For singing, the low pitch was produced in the chest register and the high notes in the head register. We collected the data on the long-term average spectra of the speaking and singing voices of the trained singers and the control groups.ResultsFor the sounds produced from the head register, a significant energy concentration was seen in both 2.2-3.4 kHz and 7.5-8.4 kHz regions (except for the voices of the mezzo sopranos) in the trained singer group when compared to the control groups. Also, the chest register had a significant energy concentration in the 4 trained singer groups at the 2.2-3.1 kHz and 7.8-8.4 kHz. For speaking sound, all trained singers had a significant energy concentration at 2.2-5.3 kHz and sopranos had another energy concentration at 9-10 kHz.ConclusionThe results of this study suggest that opera singers have more energy concentration in the singer's formant/speaker's ring region, in both singing and speaking voices. Furthermore, another region of energy concentration was identified in opera singer's singing sound and in sopranos' speaking sound at 8-9 kHz. The authors believe that these energy concentrations may contribute to the rich voice of trained singers

Učinci prenatalne i postnatalne izloženosti perfluorooktanskoj kiselini na ekspresiju glavnih gena povezanih s reprodukcijom u mišjem hipotalamusu i spolnim žlijezdama

Perfluorooctanoic acid (PFOA), a ubiquitous environmental pollutant reported to be an endocrine disruptor, is used in many industrial and consumer products. Although the adverse effects of PFOA on the reproductive health of animals and humans have been widely reported, most studies have focused on assessing the anatomical features and conventional histology of adult gonads. Therefore, the molecular mechanisms activated in the hypothalamus and gonads following PFOA exposure during the pre- and postnatal periods are not clear. This study used a mouse model to evaluate the effects of PFOA exposure on the alteration of molecular mechanisms in the hypothalamus and gonads during the prenatal and postpartum periods. Changes in gene and protein expression following PFOA exposure were evaluated by quantitative polymerase chain reaction and Western blotting, respectively. Kisspeptin 1 and gonadotropin-releasing hormone expression in the hypothalamus of female and male mouse pups was significantly decreased. Additionally, Cyp17a1 expression was upregulated in male offspring testes, while Cyp17a1 and Cyp19a1 expression was downregulated in female offspring ovaries. Changes at the molecular level due to PFOA exposure in the early stages of development did not show sex-related differences in the hypothalamus; however, such differences were confirmed in the gonads. These results could be used as basic data to study the molecular mechanisms underlying the reproductive dysfunction caused by PFOA exposure in the early stages of embryonic development.Perfluorooktanska kiselina (PFOA) sveprisutna je onečišćujuća tvar za okoliš, za koju je zabilježeno da uzrokuje i endokrinopatije, a upotrebljava se u mnogim industrijskim proizvodima. Bez obzira na poznate i zabilježene nuspojave PFOA-e na reproduktivno zdravlje životinja i ljudi, većina se istraživanja usredotočuje na procjenu anatomskih histoloških značajki u spolnim žlijezdama odraslih jedinki. Molekularni mehanizmi koji se aktiviraju u hipotalamusu i spolnim žlijezdama nakon izlaganja PFOA-i stoga nisu razjašnjeni. U ovom je istraživanju upotrijebljen mišji model za procjenu učinaka izlaganja PFOA-i na promjenu molekularnih mehanizama u hipotalamusu i spolnim žlijezdama za vrijeme prijeporođajnog i poslijeporođajnog razdoblja. Promjene u ekspresiji gena i proteina nakon izloženosti PFOA-i analizirane su kvantitativnom PCR metodom i metodom Western blotting. Ekspresija kispeptina 1 i hormona koji oslobađa gonadotropin u hipotalamusu ženske i muške mladunčadi bila je znakovito smanjena. Osim toga ekpresija Cyp17a1 bila je pojačana u testisima muških potomaka, dok je ekspresija Cyp17a1 i Cyp19a1 u jajnicima ženskih potomaka bila smanjena. Kod promjena na molekularnoj razini u hipotalamusu u ranim razvojnim stadijima nije bilo razlike među spolovima, dok je kod promjena u spolnim žlijezdama povrđena razlika među spolovima. Rezultati ovog istraživanja mogli bi biti korisni kao osnovni podaci u proučavanju molekularnih mehanizama podležeće reproduktivne disfunkcije uzrokovane izloženošću PFOA-i u ranim stadijima embrionalnog razvoja

Characterization of GDP-mannose Pyrophosphorylase from Escherichia Coli O157:H7 EDL933 and Its Broad Substrate Specificity

GDP-mannose pyrophosphorylase gene (ManC) of Escherichia coli (E. coli) O157 was cloned and expressed as a highly soluble protein in E. coli BL21 (DE3). The enzyme was subsequently purified using hydrophobic and ion exchange chromatographies. ManC showed very broad substrate specificities for four nucleotides and various hexose-1-phosphates, yielding ADP-mannose, CDP-mannose, UDP-mannose, GDP-mannose, GDP-glucose and GDP-2-deoxy-glucose

Heterologous Expression of Plant Cell Wall Degrading Enzymes for Effective Production of Cellulosic Biofuels

A major technical challenge in the cost-effective production of cellulosic biofuel is the need to lower the cost of plant cell wall degrading enzymes (PCDE), which is required for the production of sugars from biomass. Several competitive, low-cost technologies have been developed to produce PCDE in different host organisms such as Escherichia coli, Zymomonas mobilis, and plant. Selection of an ideal host organism is very important, because each host organism has its own unique features. Synthetic biology-aided tools enable heterologous expression of PCDE in recombinant E. coli or Z. mobilis and allow successful consolidated bioprocessing (CBP) in these microorganisms. In-planta expression provides an opportunity to simplify the process of enzyme production and plant biomass processing and leads to self-deconstruction of plant cell walls. Although the future of currently available technologies is difficult to predict, a complete and viable platform will most likely be available through the integration of the existing approaches with the development of breakthrough technologies.open8

Computational models for the ultimate compressive strength analysis of a steel stiffened plate structure at cryogenic condition

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Effects of Wet-Pressing and Cross-Linking on the Tensile Properties of Carbon Nanotube Fibers

To increase the strength of carbon nanotube (CNT) fibers (CNTFs), the mean size of voids between bundles of CNTs was reduced by wet-pressing, and the CNTs were cross-linked. Separate and simultaneous physical (roller pressing) and chemical methods (cross-linking) were tested to confirm each method's effects on the CNTF strength. By reducing the fraction of pores, roller pressing decreased the cross-sectional area from 160 mu m(2) to 66 mu m(2) and increased the average load-at-break from 2.83 +/- 0.25 cN to 4.41 +/- 0.16 cN. Simultaneous injection of crosslinker and roller pressing augmented the cross-linking effect by increasing the infiltration of the crosslinker solution into the CNTF, so the specific strength increased from 0.40 +/- 0.05 N/tex to 0.67 +/- 0.04 N/tex. To increase the strength by cross-linking, it was necessary that the size of the pores inside the CNTF were reduced, and the infiltration of the solution was increased. These results suggest that combined physical and chemical treatment is effective to increase the strength of CNTFs.11Ysciescopu