Free Radical Chemistry of Phosphasilenes

Understanding the characteristics of radicals formed from silicon-containing heavy analogues of alkenes is of great importance for their application in radical polymerization. Bulky and electronic substituent effects in such compounds as phosphasilenes not only stabilize the Si=P double bond, but also influence the structure and species of the formed radicals. Herein we report our first investigations of radicals derived from phosphasilenes with Mes (2,4,6-trimethylphenyl), Tip (2,4,6-triisopropylphenyl), Dur (2,3,5,6-tetramethylphenyl) and NMe2 (dimethylamino) substituents on the P atom, using muon spin spectroscopy and DFT calculations. Adding muonium (a light isotope of hydrogen) to phosphasilenes reveals that: a) the electron-donor NMe2 and the bulkiest Tip-substituted phosphasilenes form several muoniated radicals with different rotamer conformations; b) bulky Dur-substituted phosphasilene forms two radicals (Si- and P-centred); and c) Mes-substituted phosphasilene mainly forms one species of radical, at the P centre. These significant differences result from intramolecular substituent effects

Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation.

Adult somatic tissues have proven difficult to expand in vitro, largely because of the complexity of recreating appropriate environmental signals in culture. We have overcome this problem recently and developed culture conditions for adult stem cells that allow the long-term expansion of adult primary tissues from small intestine, stomach, liver and pancreas into self-assembling 3D structures that we have termed 'organoids'. We provide a detailed protocol that describes how to grow adult mouse and human liver and pancreas organoids, from cell isolation and long-term expansion to genetic manipulation in vitro. Liver and pancreas cells grow in a gel-based extracellular matrix (ECM) and a defined medium. The cells can self-organize into organoids that self-renew in vitro while retaining their tissue-of-origin commitment, genetic stability and potential to differentiate into functional cells in vitro (hepatocytes) and in vivo (hepatocytes and endocrine cells). Genetic modification of these organoids opens up avenues for the manipulation of adult stem cells in vitro, which could facilitate the study of human biology and allow gene correction for regenerative medicine purposes. The complete protocol takes 1-4 weeks to generate self-renewing 3D organoids and to perform genetic manipulation experiments. Personnel with basic scientific training can conduct this protocol.LB is supported by an EMBO Postdoctoral fellowship (EMBO ALTF 794-2014). CH is supported by a Cambridge Stem Cell Institute Seed Fund award and the Herchel Smith Fund. BK is supported by a Sir Henry Dale Fellowship from the Wellcome Trust and the Royal Society. MH is a Wellcome Trust Sir Henry Dale Fellow and is jointly funded by the Wellcome Trust and the Royal Society (104151/Z/14/Z).This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/nprot.2016.097

Chemie freier Radikale von Phosphasilenen

Das Verständnis der Eigenschaften von Radikalen, die ausgehend von Si-haltigen, schweren Analoga von Alkenen gebildet werden, ist wichtig für ihre Anwendung in der radikalischen Polymerisation. Sterische und elektronische Substituenteneffekte in Phosphasilenen stabilisieren nicht nur die Si=P-Doppelbindung, sondern beeinflussen auch die Struktur und Natur der gebildeten Radikale. Wir berichten hier über Untersuchungen an Phosphasilen-abgeleiteten Radikalen mit Mes-, Tip-, Dur- und NMe2-Substituenten am P-Atom mithilfe von Myonenspinspektroskopie und DFT-Rechnungen. Die Addition von Myonium (einem leichten Isotop von Wasserstoff) an Phosphasilene zeigt, dass a) das Elektronendonor-NMe2- und das sperrigste Tip-substituierte Phosphasilen mehrere myonierte Radikale mit unterschiedlichen Rotamer-Konformationen bilden; b) das sperrige Dur-substituierte Phosphasilen zwei Radikale (Si- und P-zentriert) bildet; und c) Mes-substituiertes Phosphasilen hauptsächlich eine Radikalspezies am P-Zentrum bildet. Diese signifikanten Unterschiede rühren von einem intramolekularen Substituenteneffekt her

Micro-computed tomography (μ-CT) as a potential tool to assess the effect of dynamic coating routes on the formation of biomimetic apatite layers on 3D-plotted biodegradable polymeric scaffolds

This work studies the influence of dynamic biomimetic coating procedures on the growth of bonelike apatite layers at the surface of starch/polycaprolactone (SPCL) scaffolds produced by a 3D-plotting technology. These systems are newly proposed for bone Tissue Engineering applications. After generating stable apatite layers through a sodium silicate-based biomimetic methodology the scaffolds were immersed in Simulated Body Fluid solutions (SBF) under static, agitation and circulating flow perfusion conditions, for different time periods. Besides the typical characterization techniques, Micro-Computed Tomography analysis (μ-CT) was used to assess scaffold porosity and as a new tool for mapping apatite content. 2D histomorphometric analysis was performed and 3D virtual models were created using specific softwares for CT reconstruction. By the proposed biomimetic routes apatite layers were produced covering the interior of the scaffolds, without compromising their overall morphology and interconnectivity. Dynamic conditions allowed for the production of thicker apatite layers as consequence of higher mineralizing rates, when comparing with static conditions. μ-CT analysis clearly demonstrated that flow perfusion was the most effective condition in order to obtain well-defined apatite layers in the inner parts of the scaffolds. Together with SEM, this technique was a useful complementary tool for assessing the apatite content in a non-destructive way

ISSCR standards for the use of human stem cells in basic research.

The laboratory culture of human stem cells seeks to capture a cellular state as an in vitro surrogate of a biological system. For the results and outputs from this research to be accurate, meaningful, and durable, standards that ensure reproducibility and reliability of the data should be applied. Although such standards have been previously proposed for repositories and distribution centers, no widely accepted best practices exist for laboratory research with human pluripotent and tissue stem cells. To fill that void, the International Society for Stem Cell Research has developed a set of recommendations, including reporting criteria, for scientists in basic research laboratories. These criteria are designed to be technically and financially feasible and, when implemented, enhance the reproducibility and rigor of stem cell research

Liver cell therapy: is this the end of the beginning?

The prevalence of liver diseases is increasing globally. Orthotopic liver transplantation is widely used to treat liver disease upon organ failure. The complexity of this procedure and finite numbers of healthy organ donors have prompted research into alternative therapeutic options to treat liver disease. This includes the transplantation of liver cells to promote regeneration. While successful, the routine supply of good quality human liver cells is limited. Therefore, renewable and scalable sources of these cells are sought. Liver progenitor and pluripotent stem cells offer potential cell sources that could be used clinically. This review discusses recent approaches in liver cell transplantation and requirements to improve the process, with the ultimate goal being efficient organ regeneration. We also discuss the potential off-target effects of cell-based therapies, and the advantages and drawbacks of current pre-clinical animal models used to study organ senescence, repopulation and regeneration

Membrane-association of mRNA decapping factors is independent of stress in budding yeast

Recent evidence has suggested that the degradation of mRNA occurs on translating ribosomes or alternatively within RNA granules called P bodies, which are aggregates whose core constituents are mRNA decay proteins and RNA. In this study, we examined the mRNA decapping proteins, Dcp1, Dcp2, and Dhh1, using subcellular fractionation. We found that decapping factors co-sediment in the polysome fraction of a sucrose gradient and do not alter their behaviour with stress, inhibition of translation or inhibition of the P body formation. Importantly, their localisation to the polysome fraction is independent of the RNA, suggesting that these factors may be constitutively localised to the polysome. Conversely, polysomal and post-polysomal sedimentation of the decapping proteins was abolished with the addition of a detergent, which shifts the factors to the non-translating RNP fraction and is consistent with membrane association. Using a membrane flotation assay, we observed the mRNA decapping factors in the lower density fractions at the buoyant density of membrane-associated proteins. These observations provide further evidence that mRNA decapping factors interact with subcellular membranes, and we suggest a model in which the mRNA decapping factors interact with membranes to facilitate regulation of mRNA degradation

A systematic review and meta-analysis to determine the contribution of mr imaging to the diagnosis of foetal brain abnormalities In Utero.

OBJECTIVES: This systematic review was undertaken to define the diagnostic performance of in utero MR (iuMR) imaging when attempting to confirm, exclude or provide additional information compared with the information provided by prenatal ultrasound scans (USS) when there is a suspicion of foetal brain abnormality. METHODS: Electronic databases were searched as well as relevant journals and conference proceedings. Reference lists of applicable studies were also explored. Data extraction was conducted by two reviewers independently to identify relevant studies for inclusion in the review. Inclusion criteria were original research that reported the findings of prenatal USS and iuMR imaging and findings in terms of accuracy as judged by an outcome reference diagnosis for foetal brain abnormalities. RESULTS: 34 studies met the inclusion criteria which allowed diagnostic accuracy to be calculated in 959 cases, all of which had an outcome reference diagnosis determined by postnatal imaging, surgery or autopsy. iuMR imaging gave the correct diagnosis in 91 % which was an increase of 16 % above that achieved by USS alone. CONCLUSION: iuMR imaging makes a significant contribution to the diagnosis of foetal brain abnormalities, increasing the diagnostic accuracy achievable by USS alone. KEY POINTS: • Ultrasound is the primary modality for monitoring foetal brain development during pregnancy • iuMRI used together with ultrasound is more accurate for detecting foetal brain abnormalities • iuMR imaging is most helpful for detecting midline brain abnormalities • The moderate heterogeneity of reviewed studies may compromise findings