Isolation and culture of adult intestinal, gastric, and lver organoids for cre-recombinase-mediated gene deletion

The discovery of Lgr5 as a marker of adult stem cells meant that stem cell populations could be purified and studied in isolation. Importantly, when cultured under the appropriate conditions these stem cells form organoids in tissue culture that retain many features of the tissue of origin. The organoid cultures are accessible to genetic and biochemical manipulation, bridging the gap between in vivo mouse models and conventional tissue culture. Here we describe robust protocols to establish organoids from gastrointestinal tissues (stomach, intestine, liver) and Cre-recombinase mediated gene manipulation in vitro

Reactivity of Phenylacetylene toward Unsymmetrical Disilenes: Regiodivergent [2+2] Cycloaddition vs. CH Addition

We report the regiodivergent reaction of phenylacetylene with a selection of disilenes Tip2Si=SiTipR as well as bridged tetrasiladienes Tip2Si=SiTip−LU−SiTip=SiTip2 (Tip=2,4,6-iPr3C6H2, R=aryl groups; LU=arylene linkers). The regioselectivity of the [2+2] cycloaddition as determined by NMR spectroscopy and X-ray crystallography is shown to strongly depend on the nature of the substituent R. The small size of the substituents compared to the Tip groups in both cases suggests a change in mechanism between the substrates with only hydrogen in the ortho-positions of R and LU and those with either ortho-methyl groups or condensed aromatic rings. In contrast, the presence of catalytic quantities of base completely suppresses cycloaddtion in favor of the formal CH addition of phenylacetylene. Quenching reactions with either MeI or MeOH after the stoichiometric application of deprotonated phenylacetylene as well as NMR studies at low temperature prove the intermediacy of an alkynyl-substituted disilanyl lithium and thus suggest a carbolithiation pathway for the net CH addition

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

Equilibrium Formation of Stable All‐Silicon Versions of 1,3‐Cyclobutanediyl

Main group analogues of cyclobutane‐1,3‐diyls are fascinating due to their unique reactivity and electronic properties. So far only heteronuclear examples have been isolated. Here we report the isolation and characterization of all‐silicon 1,3‐cyclobutanediyls as stable closed‐shell singlet species from the reversible reactions of cyclotrisilene c ‐Si3Tip4 (Tip=2,4,6‐triisopropylphenyl) with the N‐heterocyclic silylenes c ‐[(CR2CH2)(Nt Bu)2]Si: (R=H or methyl) with saturated backbones. At elevated temperatures, tetrasilacyclobutenes are obtained from these equilibrium mixtures. The corresponding reaction with the unsaturated N‐heterocyclic silylene c ‐(CH)2(Nt Bu)2Si: proceeds directly to the corresponding tetrasilacyclobutene without detection of the assumed 1,3‐cyclobutanediyl intermediate

k-Universality of Regular Languages

A subsequence of a word w is a word u such that u = w[i1]w[i2] . . . w[ik], for some set of indices 1 ≤ i1 < i2 < · · · < ik ≤ |w|. A word w is k-subsequence universal over an alphabet Σ if every word in Σk appears in w as a subsequence. In this paper, we study the intersection between the set of k-subsequence universal words over some alphabet Σ and regular languages over Σ. We call a regular language L k-∃-subsequence universal if there exists a k-subsequence universal word in L, and k-∀-subsequence universal if every word of L is k-subsequence universal. We give algorithms solving the problems of deciding if a given regular language, represented by a finite automaton recognising it, is k-∃-subsequence universal and, respectively, if it is k-∀-subsequence universal, for a given k. The algorithms are FPT w.r.t. the size of the input alphabet, and their run-time does not depend on k; they run in polynomial time in the number n of states of the input automaton when the size of the input alphabet is O(log n). Moreover, we show that the problem of deciding if a given regular language is k-∃-subsequence universal is NP-complete, when the language is over a large alphabet. Further, we provide algorithms for counting the number of k-subsequence universal words (paths) accepted by a given deterministic (respectively, nondeterministic) finite automaton, and ranking an input word (path) within the set of k-subsequence universal words accepted by a given finite automaton

MRI of female genital tract congenital anomalies: European Society of Urogenital Radiology (ESUR) guidelines

OBJECTIVE: To develop imaging guidelines for the MR work-up of female genital tract congenital anomalies (FGTCA). METHODS: These guidelines were prepared based on a questionnaire sent to all members of the European Society of Urogenital Radiology (ESUR) Female Pelvic Imaging Working Group (FPI-WG), critical review of the literature and expert consensus decision. RESULTS: The returned questionnaires from 17 different institutions have shown reasonable homogeneity of practice. Recommendations with focus on patient preparation and MR protocol are proposed, as these are key to optimised examinations. Details on MR sequences and planning of uterus-orientated sequences are provided. CONCLUSIONS: The multiplanar capabilities and soft tissue resolution of MRI provide superb characterisation of the wide spectrum of findings in FGTCA. A standardised imaging protocol and method of reporting ensures that the salient features are recognised, contributing to a correct diagnosis and classification of FGTCA, associated anomalies and complications. These imaging guidelines are based on current practice among expert radiologists in the field and incorporate up to date information regarding MR protocols and essentials of recently published classification systems. KEY POINTS: * MRI allows comprehensive evaluation of female genital tract congenital anomalies, in a single examination. * A dedicated MRI protocol comprises uterus-orientated sequences and vaginal and renal evaluation. * Integration of classification systems and structured reporting helps in successful communication of the imaging findings

Five years’ trajectories of functionality and pain in patients after hip or knee replacement and association with long-term patient survival

To describe the 5 years’ trajectories in functionality and pain of patients with hip or knee osteoarthritis and arthroplasty and analyze the association of these with long-term patients survival. Patients with OA receiving total hip or knee arthroplasty were recruited and completed two sets of standardized questionnaires for functionality and pain 6, 12, and 60 months postoperatively. Multivariate mixed models were conducted to assess trajectories over time and the resulting improvement per month during the last time period was included in a landmark-model to estimate adjusted hazard ratios for mortality. In total 809 patients with joint replacement were included (mean age 65.0 years, 62.2% female), 407 patients died (median follow-up 18.4 years). Both instruments of functionality and pain showed extensive improvement during the first 6 months. Baseline and change in functionality (both p < 0.001) and pain (p = 0.02) during the first 6 months were associated with mortality. Better values in functionality corresponded with improved survival whereas the association with the pain scores was inverse. In patients with hip and knee OA, an explicit improvement in function is seen within the first 6 months after arthroplasty. In addition, especially the functionality scores at baseline as well as their improvement showed an association with long-term patient survival

Test, Reliability and Functional Safety Trends for Automotive System-on-Chip

This paper encompasses three contributions by industry professionals and university researchers. The contributions describe different trends in automotive products, including both manufacturing test and run-time reliability strategies. The subjects considered in this session deal with critical factors, from optimizing the final test before shipment to market to in-field reliability during operative life

Bildung Stabiler All‐Silicium Varianten von 1,3‐Cyclobutandiyl im Gleichgewicht

Hauptgruppenanaloga von 1,3‐Cyclobutandiylen faszinieren mit ihrer einzigartigen Reaktivität und ihren elektronischen Eigenschaften. Bisher sind allerdings nur heteronukleare Vertreter isoliert worden. Wir berichten hier über die Isolierung und Charakterisierung von All‐Silicium‐1,3‐Cyclobutandiylen als stabile Singulettspezies mit geschlossenschaliger Konfiguration aus den reversiblen Reaktionen von Cyclotrisilen c ‐Si3Tip4 (Tip=2,4,6‐Triisopropylphenyl) mit den N‐heterocyclischen Silylenen c ‐[(CR2CH2)(Nt Bu)2]Si: (R=H oder Methyl) mit gesättigten Grundgerüsten. Bei erhöhten Temperaturen werden aus diesen Gleichgewichtsmischungen Tetrasilacyclobutene erhalten. Die analoge Reaktion mit dem ungesättigten N‐heterocyclischen Silylen c ‐(CH)2(Nt Bu)2Si: verläuft direkt zum entsprechenden Tetrasilacyclobuten ohne Nachweis des angenommenen 1,3‐Cyclobutandiyl‐Zwischenprodukts