Regulation of trophoblast stem cell maintenance and differentiation by LIN28 and AP-2γ

2010 Summer.Includes bibliographic references (pages 89-96).Covers not scanned.Print version deaccessioned 2022.The placenta is a unique organ essential for survival of the fetus in all eutherian mammals. Failure to develop a normal placenta in humans can lead to diseases, such as pre-eclampsia, with high morbidity and mortality for both the mother and the fetus. These diseases are thought to be caused by abnormal proliferation and differentiation of cells in the placenta. A mouse trophoblast stem (TS) cell culture system is a useful tool in studying TS cell proliferation and differentiation into trophoblast giant cells (TGCs). TS cells cultured in proliferative media (70% conditioned media, 30% TS media, FGF4, and heparin sulfate) will remain proliferative, and TS cells cultured under differentiation media (100% TS media) will differentiate into TGCs. LIN28 is a protein that regulates mRNAs and miRNAs, and is abundantly expressed in many undifferentiated tissues. AP- 2y has been shown to be essential for TS cell maintenance and TGC formation. AP-2y null mutants display embryonic lethality at E7.5 due to a severely disrupted extraembryonic portion of the embryo. In TS cells, AP-2y has been shown to bind to the promoter region of Lin28. This study investigates the hypothesis that Liti28 and Ap-2y are necessary regulators of trophoblast stem cell maintenance and differentiation into TGCs. This study shows the pluripotency genes, Lin28, Sox2, and NrObl, to be differentially expressed in proliferating TS cells and differentiated TGCs. MiRNAs can be used as markers for proliferation or differentiation. 28 significantly different miRNAs were detected between TS cells and TGCs, 18 up-regulated in TGCs and 9 downregulated in TGCs. Expression of the miR-290 family, initially thought to be ES cell specific, was detected in proliferating TS cells suggesting TS cells have similar miRNA mediated regulation of proliferation compared to ES cells. The Let-7 family of miRNAs was found to be up-regulated in differentiated TGCs. The Let-7 family has been shown to be regulated by LIN28, where LIN28 prevents accumulation of mature Let-7 miRNAs. In this study Lin28 was highly expressed in proliferating cells and the Let-7’s are upregulated in differentiated TGCs. Lin28 function in TS cells was assessed by knocking down Lin28 using shRNA lentiviral technology. Lin28 knockdown TS cells were used to observe results of knockdown. We obtained a 78% reduction of Lin28 mRNA, but found that loss of Lin28 in TS cells did not affect morphology, proliferation or differentiation. AP-2y null TS cells grown in culture fail to differentiate morphologically into TGCs. Lin28, Sox2, and NrObl show no difference in expression when grown in conditions to differentiate the cells, indicating a failure of AP-2y null TS cells to differentiate into TGCs. RO3306 is a compound used to block Cyclin-dependent Protein Kinase 1 and force endoreduplication, causing TS cells to differentiate into TGCs. AP-2y null TS cells cannot be forced to differentiate into TGCs, and instead undergo cell death, when cultured with RO3306. Additionally, AP-2y null TS cells express the pluripotency markers Oct4, Stella, and Nanog which only are expressed in ES cells and germ cells. MiRNA profiling of AP-2y null TS cells indicates that cells in proliferative conditions resemble wild type counterparts, but when proliferative conditions are removed we observe an increase in expression of the ES cell specific miR-302 cluster. While there was no effect of proliferation in wild type cells, loss of Lin28 in AP-2y null TS cells via lentiviral knockdown leads to a partial rescue of TGC formation. This suggests that Liii28 must be down-regulated in order for TGC formation, and that AP-2y regulates Lin28 in TS cells. Taken together these data suggest a role for Lin28 in mouse TS cell proliferation and differentiation, where Lin28 must become down regulated in order for differentiation into TGCs. AP-2y has been shown to bind to the Lin28 promoter in TS cells; this regulation enables TS cell differentiation into TGCs. This study also shows the necessity of AP-2y for TS cell differentiation into TGCs; loss of AP-2y leads to a more pluripotent state rather than allowing for differentiation. Loss of AP-2y leads to expression of pluripotency markers Oct4, Nanog, and Stella, and the ES cell specific miR-302 cluster, indicating an increase in pluripotency. We conclude that AP-2y and LIN28 are essential molecular regulators of TS cell proliferation and differentiation

Edge-illumination X-ray dark-field imaging for visualising defects in composite structures

Low velocity impact can lead to barely visible and difficult to detect damage such as fibre and matrix breakage or delaminations in composite structures. Drop-weight impact damage in a cross-ply carbon fibre laminate plate was characterized using ultrasonic C-scan measurements. This was compared to the results provided by a novel X-ray imaging technique based on the detection of phase effects, which can be implemented with conventional equipment. Three representations of the sample are provided: absorption, differential phase and dark-field. The latter is of particular interest to detect cracks and voids of dimensions that are smaller than the spatial resolution of the imaging system. The ultrasonic C-scan showed a large delamination and additional damage along the fibre directions. The damage along the fibre directions and other small scale defects were detected from the X-ray imaging. As the system is sensitive to phase effects along one direction at a time, the acquisition of an additional scan, rotating the sample 90 degrees around the beam axis, provides information in both fibre directions. These two techniques enable access to a set of complementary information, across different length scales, which can be useful in the characterization of the defects occurring in composite structures

THE EFFECTS OF ANKLE BRACING ON SPORTS-SPECIFIC CAPABILITIES

INTRODUCTION Ankle braces are widely used in athletes. In some professional sports leagues it is mandatory to wear ankle braces in order to get insurance. Even prophylactic bracing is often used in normal sports. The efficiency of ankle joint orthoses was already demonstrated in several studies, and different types of external stabilization devices were compared. Especially the. aspects of mechanical stabilization were discussed in these studies as well as retrospective observations of the prophylactic benefit regarding the prevention of sprains by using orthoses. Neurophysiological or psychological aspects are hardly taken into consideration in the literature when evaluating different types of orthoses. In the presented study the proprioception with regard to the ankle joint and especially its influence by orthoses and the effect of ankle braces on sports- specific capabilities will be discussed. First of all the question is, whether the orthoses have an impact on the proprioception of the ankle joint at all. If there is an influence, then it is of particular importance for a comprehensive judgement to document the degree of influence by each type of orthoses for the injured and not injured ankle joint. By evaluating the results of this study a possible reduction of the proprioception caused by ankle sprains will be investigated as well. This phenomenon of a posttraumatic proprioceptive deficit was already described and controversially discussed in the literature. MATERIALS AND METHODS The influence of four stabilizing devices (aircast brace, . ligafix air-brace, malleoloc-brace, taping) on sportsspecific capabilities and propioceptivity of stable and unstable ankle joints were assessed. Therefore both ankle joints of 18 uninjured volunteers and 23 injured volunteers with an old ankle sprain were tested with two different set-ups (modified Japan-test, 5 point single leg jump test). We used a modular coordination-testsystem with 5 contact plates and a computer monitor that gives in randomised sequence the signal which circles of the 5 plates to jump on. RESULTS The time for both tests as well as reaction time and contact time for each moving direction was measured and calculated by a software program. Regarding the modified Japan-test and the 5 point single leg jump test the injured group achieved showed significant better results (p< 0.01) & (

Partial Bone Formation in Additive Manufactured Porous Implants Reduces Predicted Stress and Danger of Fatigue Failure

New porous implant designs made possible by additive manufacturing allow for increased osseointegration, potentially improving implant performance and longevity for patients that require massive bone implants. The aim of this study was to evaluate how implantation and the strain distribution in the implant affect the pattern of bone ingrowth and how changes in tissue density within the pores alter the stresses in implants. The hypothesis was that porous metal implants are susceptible to fatigue failure, and that this reduces as osteointegration occurs. A phenomenological, finite element analysis (FEA) bone remodelling model was used to predict partial bone formation for two porous (pore sizes of 700 μm and 1500 μm), laser sintered Ti_{6}Al_{4}V implants in an ovine condylar defect model, and was compared and verified against in vivo, histology results. The FEA models predicted partial bone formation within the porous implants, but over-estimated the amount of bone-surface area compared to histology results. The stress and strain in the implant and adjacent tissues were assessed before, during bone remodelling, and at equilibrium. Results showed that partial bone formation improves the stress distribution locally by reducing stress concentrations for both pore sizes, by at least 20%. This improves the long-term fatigue resistance for the larger pore implant, as excessively high stress is reduced to safer levels (86% of fatigue strength) as bone forms. The stress distribution only changed slightly in regions without bone growth. As the extent of bone formation into extensively porous bone implants depends on the level of stress shielding, the design of the implant and stiffness have significant influence on bone integration and need to be considered carefully to ensure the safety of implants with substantial porous regions. To our knowledge this is the first time that the effect of bone formation on stress distribution within a porous implant has been described and characterised

Post-Acquisition Mask Misalignment Correction for Edge Illumination X-ray Phase Contrast Imaging

Edge illumination x-ray phase contrast imaging uses a set of apertured masks to translate phase effects into variation of detected intensity. While the system is relatively robust against misalignment, mask movement during acquisition can lead to gradient artifacts. A method has been developed to correct the images by quantifying the misalignment post-acquisition and implementing correction maps to remove the gradient artifact. Images of a woven carbon fiber composite plate containing porosity were used as examples to demonstrate the image correction process. The gradient formed during image acquisition was removed without affecting the image quality, and results were subsequently used for quantification of porosity, indicating that the gradient correction did not affect the quantitative content of the images

Bone remodeling in additive manufactured porous implants changes the stress distribution

Safety and efficacy of additive manufactured porous implants is a growing concern due to several, recent recalls. The safety of bone implants depends on the effects of implantation and partial bone ingrowth on stress and strain. Finite element analysis, using two new algorithms to simulate bone ingrowth, was verified against histology results for an ovine condylar critical sized defect model. Implants were manufactured from Ti6Al4V using selective laser sintering. Results showed that partial bone formation reduces stress concentrations to safe levels, improving the long-term fatigue resistance. Higher bone ingrowth was predicted for implants made from lower modulus Titanium-tantalum alloy

EFFECT OF DIFFERENT LOW BACK TRAINING PROGRAMS ON LUMBAR SPINE KINESTHESIA

Reduced kinesthetic perceptions can impair lower back sensorimotor functions and result in increased injury risk. The effect of low back training programs on lumbar spine kinesthetic sensibility is undetermined. There was a back strengthening exercise group (with low back pain; training 4.4 h/wk), a “classical” back training program group (with low back pain; training 4.9 h/wk) and a control group (training 5.4 h/wk). During an active reproduction test, subjects performed trunk positions in random order: flexion [A(0°-20°), B(20°-40°)], lateral flexion [C(0°-30°)], Using a 3D-ultrasound motion analysis system the repositioning error was calculated from the given target position to the subject perceived target position, before and after a 5 week training period. Results show decreased repositioning error after the training for both training groups

Electroweak Baryogenesis in Non-minimal Composite Higgs Models

We address electroweak baryogenesis in the context of composite Higgs models, pointing out that modifications to the Higgs and top quark sectors can play an important role in generating the baryon asymmetry. Our main observation is that composite Higgs models that include a light, gauge singlet scalar in the spectrum [as in the model based on the symmetry breaking pattern SO(6)/SO(5)], provide all necessary ingredients for viable baryogenesis. In particular, the singlet leads to a strongly first-order electroweak phase transition and introduces new sources of CP violation in dimension-five operators involving the top quark. We discuss the amount of baryon asymmetry produced and the experimental constraints on the model.Comment: 15 pages, 7 figure

Quantification of porosity in composite plates using planar X-ray phase contrast imaging

The application of planar Edge-Illumination X-ray Phase-Contrast imaging (EI-XPCi) for the non-destructive quantification of porosity in carbon fiber reinforced polymer (CFRP) specimens, a significant concern in aerospace applications, was investigated. The method enables fast, planar (2D) scans providing access to large samples. A set of woven CFRP plates with porosity content ranging from 0.7% to 10.7% was examined. In addition to standard X-ray attenuation, EI-XPCi provides differential phase and dark-field signals, sensitive to inhomogeneities and interfaces at scales above and below the system spatial resolution, respectively. The correlation with the porosity content from matrix digestion obtained from the dark-field signal was comparable to that from ultrasonic attenuation. The novel analysis of the standard deviation of differential phase (STDP), sensitive to inhomogeneities above the system resolution (approximately 12 μm), resulted in a very high correlation (R2 = 0.995) with the matrix digestion porosity content, outperforming ultrasonic attenuation measurements