3D-Image analysis platform monitoring relocation of pluripotency genes during reprogramming

Nuclear organization of chromatin is an important level of genome regulation with positional changes of genes occurring during reprogramming. Inherent variability of biological specimens, wide variety of sample preparation and imaging conditions, though pose significant challenges to data analysis and comparison. Here, we describe the development of a computational image analysis toolbox overcoming biological variability hurdles by a novel single cell randomizing normalization. We performed a comparative analysis of the relationship between spatial positioning of pluripotency genes with their genomic activity and determined the degree of similarity between fibroblasts, induced pluripotent stem cells and embryonic stem cells. Our analysis revealed a preferred positioning of actively transcribed Sox2, Oct4 and Nanog away from the nuclear periphery, but not from pericentric heterochromatin. Moreover, in the silent state, we found no common nuclear localization for any of the genes. Our results suggest that the surrounding gene density hinders relocation from an internal nuclear position. Altogether, our data do not support the hypothesis that the nuclear periphery acts as a general transcriptional silencer, rather suggesting that internal nuclear localization is compatible with expression in pluripotent cells but not sufficient for expression in mouse embryonic fibroblasts. Thus, our computational approach enables comparative analysis of topological relationships in spite of stark morphological variability typical of biological data sets

Analysis of infectious virus clones from two HIV-1 superinfection cases suggests that the primary strains have lower fitness

<p>Abstract</p> <p>Background</p> <p>Two HIV-1 positive patients, L and P, participating in the Amsterdam Cohort studies acquired an HIV-1 superinfection within half a year from their primary HIV-1 infection (Jurriaans <it>et al</it>., <it>JAIDS </it>2008, <b>47:</b>69-73). The aim of this study was to compare the replicative fitness of the primary and superinfecting HIV-1 strains of both patients. The use of isolate-specific primer sets indicated that the primary and secondary strains co-exist in plasma at all time points after the moment of superinfection.</p> <p>Results</p> <p>Biological HIV-1 clones were derived from peripheral blood CD4 + T cells at different time point, and identified as the primary or secondary virus through sequence analysis. Replication competition assays were performed with selected virus pairs in PHA/IL-2 activated peripheral blood mononuclear cells (PBMC's) and analyzed with the Heteroduplex Tracking Assay (HTA) and isolate-specific PCR amplification. In both cases, we found a replicative advantage of the secondary HIV-1 strain over the primary virus. Full-length HIV-1 genomes were sequenced to find possible explanations for the difference in replication capacity. Mutations that could negatively affect viral replication were identified in the primary infecting strains. In patient L, the primary strain has two insertions in the LTR promoter, combined with a mutation in the <it>tat </it>gene that has been associated with decreased replication capacity. The primary HIV-1 strain isolated from patient P has two mutations in the LTR that have been associated with a reduced replication rate. In a luciferase assay, only the LTR from the primary virus of patient P had lower transcriptional activity compared with the superinfecting virus.</p> <p>Conclusions</p> <p>These preliminary findings suggest the interesting scenario that superinfection occurs preferentially in patients infected with a relatively attenuated HIV-1 isolate.</p

p53 Regulates Cell Cycle and MicroRNAs to Promote Differentiation of Human Embryonic Stem Cells

Multiple studies show that tumor suppressor p53 is a barrier to dedifferentiation; whether this is strictly due to repression of proliferation remains a subject of debate. Here, we show that p53 plays an active role in promoting differentiation of human embryonic stem cells (hESCs) and opposing self-renewal by regulation of specific target genes and microRNAs. In contrast to mouse embryonic stem cells, p53 in hESCs is maintained at low levels in the nucleus, albeit in a deacetylated, inactive state. In response to retinoic acid, CBP/p300 acetylates p53 at lysine 373, which leads to dissociation from E3-ubiquitin ligases HDM2 and TRIM24. Stabilized p53 binds CDKN1A to establish a G1 phase of cell cycle without activation of cell death pathways. In parallel, p53 activates expression of miR-34a and miR-145, which in turn repress stem cell factors OCT4, KLF4, LIN28A, and SOX2 and prevent backsliding to pluripotency. Induction of p53 levels is a key step: RNA-interference-mediated knockdown of p53 delays differentiation, whereas depletion of negative regulators of p53 or ectopic expression of p53 yields spontaneous differentiation of hESCs, independently of retinoic acid. Ectopic expression of p53R175H, a mutated form of p53 that does not bind DNA or regulate transcription, failed to induce differentiation. These studies underscore the importance of a p53-regulated network in determining the human stem cell state

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field

Effectiveness of a national quality improvement programme to improve survival after emergency abdominal surgery (EPOCH): a stepped-wedge cluster-randomised trial

Background: Emergency abdominal surgery is associated with poor patient outcomes. We studied the effectiveness of a national quality improvement (QI) programme to implement a care pathway to improve survival for these patients. Methods: We did a stepped-wedge cluster-randomised trial of patients aged 40 years or older undergoing emergency open major abdominal surgery. Eligible UK National Health Service (NHS) hospitals (those that had an emergency general surgical service, a substantial volume of emergency abdominal surgery cases, and contributed data to the National Emergency Laparotomy Audit) were organised into 15 geographical clusters and commenced the QI programme in a random order, based on a computer-generated random sequence, over an 85-week period with one geographical cluster commencing the intervention every 5 weeks from the second to the 16th time period. Patients were masked to the study group, but it was not possible to mask hospital staff or investigators. The primary outcome measure was mortality within 90 days of surgery. Analyses were done on an intention-to-treat basis. This study is registered with the ISRCTN registry, number ISRCTN80682973. Findings: Treatment took place between March 3, 2014, and Oct 19, 2015. 22 754 patients were assessed for elegibility. Of 15 873 eligible patients from 93 NHS hospitals, primary outcome data were analysed for 8482 patients in the usual care group and 7374 in the QI group. Eight patients in the usual care group and nine patients in the QI group were not included in the analysis because of missing primary outcome data. The primary outcome of 90-day mortality occurred in 1210 (16%) patients in the QI group compared with 1393 (16%) patients in the usual care group (HR 1·11, 0·96–1·28). Interpretation: No survival benefit was observed from this QI programme to implement a care pathway for patients undergoing emergency abdominal surgery. Future QI programmes should ensure that teams have both the time and resources needed to improve patient care. Funding: National Institute for Health Research Health Services and Delivery Research Programme

Effectiveness of a national quality improvement programme to improve survival after emergency abdominal surgery (EPOCH): a stepped-wedge cluster-randomised trial

BACKGROUND: Emergency abdominal surgery is associated with poor patient outcomes. We studied the effectiveness of a national quality improvement (QI) programme to implement a care pathway to improve survival for these patients. METHODS: We did a stepped-wedge cluster-randomised trial of patients aged 40 years or older undergoing emergency open major abdominal surgery. Eligible UK National Health Service (NHS) hospitals (those that had an emergency general surgical service, a substantial volume of emergency abdominal surgery cases, and contributed data to the National Emergency Laparotomy Audit) were organised into 15 geographical clusters and commenced the QI programme in a random order, based on a computer-generated random sequence, over an 85-week period with one geographical cluster commencing the intervention every 5 weeks from the second to the 16th time period. Patients were masked to the study group, but it was not possible to mask hospital staff or investigators. The primary outcome measure was mortality within 90 days of surgery. Analyses were done on an intention-to-treat basis. This study is registered with the ISRCTN registry, number ISRCTN80682973. FINDINGS: Treatment took place between March 3, 2014, and Oct 19, 2015. 22 754 patients were assessed for elegibility. Of 15 873 eligible patients from 93 NHS hospitals, primary outcome data were analysed for 8482 patients in the usual care group and 7374 in the QI group. Eight patients in the usual care group and nine patients in the QI group were not included in the analysis because of missing primary outcome data. The primary outcome of 90-day mortality occurred in 1210 (16%) patients in the QI group compared with 1393 (16%) patients in the usual care group (HR 1·11, 0·96-1·28). INTERPRETATION: No survival benefit was observed from this QI programme to implement a care pathway for patients undergoing emergency abdominal surgery. Future QI programmes should ensure that teams have both the time and resources needed to improve patient care. FUNDING: National Institute for Health Research Health Services and Delivery Research Programme

Rôle de la biopsie percutanée dans l’évaluation des tumeurs rénales : qu’en attendre en 2019 et quelles perspectives ?

Médecine. Anatomie et cytologie pathologiquesRésumé : les biopsies des tumeurs rénales (BR) ont montré leur intérêt en particulier en cas de découverte radiologique fortuite d’une masse rénale, bénigne dans une proportion significative de cas, et en situation métastatique, où le traitement systémique adjuvant peut être indiqué en fonction des données histopathologiques. Leur pratique s’est ainsi généralisée. Notre objectif était d’évaluer les possibilités techniques, diagnostiques et pronostiques qu’offre ce matériel restreint, afin de préciser les recommandations pour l’imageur et le pathologiste. Au total, 487 biopsies (452 patients) ont été revues. Le type tumoral, le sous-type histologique ainsi que les facteurs histo-pronostiques (grade nucléolaire ISUP (International Society of Urology Pathology), nécrose tumorale, microangioinvasion, composante sarcomatoïde/rhabdoïde et extension au tissu adipeux péri-rénal) ont été réévalués et reclassifiés si nécessaire. Un diagnostic de tumeur était possible dans 90% des cas (88% de lésions malignes, 10% bénignes et 2% de malignité incertaine). La réalisation de nombreux examens immunohistochimiques et moléculaires a été possible sur ce matériel tumoral exigu (9 mm en moyenne), permettant l’identification de tumeurs rénales rares (3,5%). Cependant, 10% des biopsies s’avéraient non informatives, en rapport avec une tumeur macroscopiquement plus petite. Parmi ces BR non informatives, certaines étaient réalisées de façon concommittante au traitement ablatif, ne permettant pas de diagnostic de lésion. Ces données ont été comparées aux pièces opératoires correspondantes lorsque celles-ci étaient accessibles (101 néphrectomies, soit 97 patients). Le caractère malin pouvait être établi sur biopsie avec une bonne sensibilité et spécificité (97% et 100%). La concordance était presque parfaite pour le type tumoral (K=0,953) et forte pour le sous-type histologique (K=0,78). Le grade ISUP était sous-estimé dans 51% des biopsies (K=0,227). L’hétérogénéité du grade, classiquement décrite sur pièces opératoires, a été relevée dans 15% des biopsies. Les autres facteurs histopronostiques présentaient une spécificité et une valeur prédictive positive élevées mais une faible sensibilité, liée à un défaut de représentation sur biopsie et/ou à une tumeur macroscopiquement plus importante. Par ailleurs, la relecture de l’ensemble des 487 biopsies a permis, à la lumière des connaissances actuelles et après réalisation éventuelle de nouveaux examens immunohistochimiques la modification de 5,5% des diagnostics initiaux (soit 26 biopsies). Notre travail souligne la fiabilité de la biopsie, devant le faible nombre de complications. La meilleure connaissance des différentes entités tumorales ainsi que de leur profil immunohistochimique et moléculaire ont permis, sur cette étude rétrospective, de préciser le diagnostic et ce, malgré la petite taille du matériel. Nos résultats illustrent également l’importance de différer la biopsie du geste thérapeutique et le besoin d’adapter les prélèvements en fonction de la taille et la présentation radiologique de la tumeur afin d’en améliorer la pertinence diagnostique et pronostique. Ces précisions apportées par la biopsie rénale s’avèrent primordiales à l’heure où l’éventail thérapeutique du cancer rénal est en pleine évolution.Summary : percutaneous renal biopsies are commonly performed for the diagnosis and management of tumor incidentally discovered on imaging or at a metastatic stage. Our study aimed to assess the technical possibilities and the diagnostic and prognostic yield that this biopsy material provides, the goal being to refine curent guidelines for radiologists and pathologists. A total of the 487 biopsies (452 patients) were reviewed. Tumor type, histological subtype and prognostic factors (lnternational Society of Urological Pathology nucleolar grade, necrosis, microscopic vascular invasion, sarcomatoid/rhabdoid component and extension into adipose tissue) have been reassessed. A tumor diagnosis was made in 90% of cases (88% malignant, 10% benign, and 2% uncertain malignancy). The small biopsy tumoral material (mean length : 9 mm) did enable to perform a number of immunohistochemical and molecular tests, allowing identification of rare kidney tumors. However, 10% of biopsies were non-diagnostic, related to a smaller tumoral macroscopic size in those patients, some of which have received concomitant ablative therapy. These datas were compared, when available (101 biopsies) to those obtained from corresponding partial or radical nephrectomy. The diagnosis ol malignancy exhibited high sensitivity and specificity on biopsy (97% and 100%). Agreement was almost perfect regarding tumor type (K=0.953), mainly renal cell carcinoma, and strong regarding histological subtype (K=0.78). ISUP grade was underestimated in 51% of the biopsies (K=0.227), with a heterogeneous pattern already observed at the level of the biopsy sample in 15% of the cases. The other histological prognostic factors showed high specificity and positive predictive value but low sensitivity owing to their insufficient quantity on the biopsy and/or to a macroscopically larger tumor. Our second reading combined with additional immunohistochemical analyses led to the reclassification of 5.5% of the diagnoses of all the 487 biopsies. Our study confirms the reliability of renal tumor biopsy, availing of additional techniques to deliver an accurate diagnosis, despite the small amount of material. lt also shows the utility to delaying biopsy in case of ablation therapy. Above all, it emphasizes the need to tailor samplin to the size and radiological characteristics of the tumor to improve the diagnostic and histological prognostic performance. As the therapeutic arsenal in kidney cancer is rapidly evolving, the place for renal biopsy appears now primordial