Development of tools for in situ hybridization in human embryonic stem cells

Abstract only availableOur laboratory uses human embryonic stem cells (hESC) to understand how embryonic cells differentiate into the cells of the placenta. The long term goal of my research is to develop in situ hybridization methods and use them to determine where genes are being expressed within differentiating hESC colonies. I focused my research on two genes that code for transcription factors, OCT 4 and GATA 2. OCT 4 is required to maintain stem cells in an undifferentiated state. GATA 2 is thought to be one of the key transcription factors that promote stem cell differentiation into syncytiotrophoblast, a placental cell type. The first step in in situ hybridization is the development of a labeled RNA probe. I designed primers to amplify a 214bp portion of the GATA2 gene sequence by PCR, and then cloned the PCR product into E. coli bacteria. I confirmed the presence and orientation of the insert by using restriction digestion and DNA sequencing. I used bacteria already transformed with a 182bp OCT4 probe sequence to create a riboprobe. I grew up the bacteria overnight, lysed them, and purified the plasmid DNA. I linearized the plasmid with a restriction enzyme, purified it, and transcribed the DNA to RNA, incorporating digoxygenin (DIG) labeled nucleotides. In the near future, I will utilize the same method to transcribe the GATA2 probe, and use both RNA probes to perform in situ hybridizations on hESC.Life Sciences Undergraduate Research Opportunity Progra

Lattice sites of ion-implanted Li in diamond

Published in: Appl. Phys. Lett. 66 (1995) 2733-2735 citations recorded in [Science Citation Index] Abstract: Radioactive Li ions were implanted into natural IIa diamonds at temperatures between 100 K and 900 K. Emission channelling patterns of a-particles emitted in the nuclear decay of 8Li (t1/2 = 838 ms) were measured and, from a comparison with calculated emission channelling and blocking effects from Monte Carlo simulations, the lattice sites taken up by the Li ions were quantitatively determined. A fraction of 40(5)% of the implanted Li ions were found to be located on tetrahedral interstitial lattice sites, and 17(5)% on substitutional sites. The fractions of implanted Li on the two lattice sites showed no change with temperature, indicating that Li diffusion does not take place within the time window of our measurements.

Vulnerability of primitive human placental trophoblast to Zika virus

Infection of pregnant women by Asian lineage strains of Zika virus (ZIKV) has been linked to brain abnormalities in their infants, yet it is uncertain when during pregnancy the human conceptus is most vulnerable to the virus. We have examined two models to study susceptibility of human placental trophoblast to ZIKV: cytotrophoblast and syncytiotrophoblast derived from placental villi at term and colonies of trophoblast differentiated from embryonic stem cells (ESC). The latter appear to be analogous to the primitive placenta formed during implantation. The cells from term placentas, which resist infection, do not express genes encoding most attachment factors implicated in ZIKV entry but do express many genes associated with antiviral defense. By contrast, the ESC-derived trophoblasts possess a wide range of attachment factors for ZIKV entry and lack components of a robust antiviral response system. These cells, particularly areas of syncytiotrophoblast within the colonies, quickly become infected, produce infectious virus and undergo lysis within 48 h after exposure to low titers (multiplicity of infection > 0.07) of an African lineage strain (MR766 Uganda: ZIKVU) considered to be benign with regards to effects on fetal development. Unexpectedly, lytic effects required significantly higher titers of the presumed more virulent FSS13025 Cambodia (ZIKVC). Our data suggest that the developing fetus might be most vulnerable to ZIKV early in the first trimester before a protective zone of mature villous trophoblast has been established. Additionally, MR766 is highly trophic toward primitive trophoblast, which may put the early conceptus of an infected mother at high risk for destruction

Analyzing Narrative Disclosure Tone in German-Language Annual Reports : A Domain-Specific Wordlist Approach

The aim of this paper is to investigate how narrative framing is utilized by HDAX companies when describing financial results. We analyzed the narratives in shareholder letters (Aktionärsbrief) and strategic reports (Lagebericht), which German companies employed to depict revenue and profit developments. In addition to focusing on negative or positive disclosure tone, as seen in previous research on English-language financial texts, we introduced an additional analysis category concerning communicated materiality, in line with the framing hypothesis of behavioral effects by users of financial reporting. Using the original texts of 89 annual reports in their native German language, we developed a domain-specific wordlists with three categories of words: (1) those used to address negative and positive developments, (2) those used to convey the manager's attitude towards reported information, and (3) those used for communicating materiality

Ultrasonic welding of lap joints of PEI plates with PEI/CF-fabric prepregs

In this study, ultrasonic welding (USW) of lap joints of polyetherimide (PEI) plates (adherends) with carbon fiber (CF) prepregs impregnated with PEI was investigated. No energy director (ED) was used, so binder contents were varied in the prepregs to compensate for the lack of the polymer in the fusion zone. In addition, the effect of the USW parameters on the structure and the mechanical properties of the lap-joints were analyzed. The most homogeneous macrostructure, the maintained structural integrity of both the CF-fabric in the prepregs and the lap-joined PEI adherends, as well as the maximum strength properties (tensile strength) were revealed for the USW joints with the minimum polymer content in the prepreg. In this case, rising the USW time from 400 up to 800 ms radically changed the macrostructure of the fusion zone, while the strength properties did not vary significantly (shear stresses were 42–48 MPa). Computer simulation of the influence of the PEI/CF-fabric ratios in the prepregs on the deformation response of the USW joints showed that the prepreg thicknesses and, accordingly, the PEI/CF ratios did not exert a noticeable effect on the strain–stress (tensile) diagrams, while the determining factor was the adhesion level

Research Notes : U.S.S.R. : Subunit composition of glycinin from various samples of cultivated and wild soybean

The major storage protein of llS class of soybean seeds, glycinin, has a complex subunit structure. Each of the six subunits is composed of two pro-tein molecules (acidic and basic), linked via disulphide bonds (Badley et al., 1975). Depending on subunit, the acidic moiety molecular weight varies from 37,000 to 42,000, with one exception (m.w. 10,000)

Ultrasonic welding of lap joints of PEI plates with PEI/CF-fabric prepregs

In this study, ultrasonic welding (USW) of lap joints of polyetherimide (PEI) plates (adherends) with carbon fiber (CF) prepregs impregnated with PEI was investigated. No energy director (ED) was used, so binder contents were varied in the prepregs to compensate for the lack of the polymer in the fusion zone. In addition, the effect of the USW parameters on the structure and the mechanical properties of the lap-joints were analyzed. The most homogeneous macrostructure, the maintained structural integrity of both the CF-fabric in the prepregs and the lap-joined PEI adherends, as well as the maximum strength properties (tensile strength) were revealed for the USW joints with the minimum polymer content in the prepreg. In this case, rising the USW time from 400 up to 800 ms radically changed the macrostructure of the fusion zone, while the strength properties did not vary significantly (shear stresses were 42�48 MPa). Computer simulation of the influence of the PEI/CF-fabric ratios in the prepregs on the deformation response of the USW joints showed that the prepreg thicknesses and, accordingly, the PEI/CF ratios did not exert a noticeable effect on the strain�stress (tensile) diagrams, while the determining factor was the adhesion leve

A six-inhibitor culture medium for improving naïve-type pluripotency of porcine pluripotent stem cells

Understanding essential signaling network requirements and making appropriate adjustments in culture conditions are crucial if porcine pluripotent stem cells (PSC) are to achieve their full potential. Here, we first used two protein factors (LIF and FGF2) and kinase inhibitor combinations in attempts to convert primed type lentiviral-reprogrammed porcine induced PSC (Lv-piPSC) into nai\u308ve-like state and developed a medium called FL6i. In addition to FGF2 and LIF, this medium contained inhibitors of MAPK14, MAPK8, TGFB1, MAP2K1, GSK3A and BMP. Crucially, the usual TGFB1 and BMP4 protein components of many stem cell media were replaced in FL6i with inhibitors of TGFB1 and BMP. With this medium, Lv-piPSC were readily transformed from their original primed state into cells that formed colonies with typical features of nai\u308ve-state stem cells. The FL6i medium also assisted generation of nai\u308ve-type piPSC lines from porcine embryonic fibroblasts with non-integrating episomal plasmids (Epi-piPSC). These lines, despite retaining variable amounts of vector DNA, expressed higher endogenous pPOU5F1 and pSOX2 than Lv-piPSC. They have been cultured without obvious morphological change for >45 passages and retained pluripotent phenotypes in terms of upregulation of genes associated with pluripotency, low expression of genes linked to emergence of somatic cell lineages, and ability to generate well differentiated teratomas in immune-compromised mice. FL6i conditions, therefore, appear to support elevated pluripotent phenotypes. However, FL6i was less able to support the generation of embryonic stem cells from porcine blastocysts. Although colonies with dome-shaped morphologies were evident and the cells had some gene expression features linked to pluripotency, the phenotypes were ultimately not stable. Pathway analysis derived from RNAseq data performed on the various cell lines generated in this study suggest the benefits of employing the FL6i medium on porcine cells reside in its ability to minimize TGFB1 and BMP signaling, which would otherwise de-stabilize the stem cell state