294 research outputs found
Engineering synucleinopathyâresistant human dopaminergic neurons by CRISPRâmediated deletion of the SNCA gene
An emerging treatment for Parkinson's disease (PD) is cell replacement therapy. Authentic midbrain dopaminergic (mDA) neuronal precursors can be differentiated from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs). These laboratoryâgenerated mDA cells have been demonstrated to mature into functional dopaminergic neurons upon transplantation into preclinical models of PD. However, clinical trials with human fetal mesenchephalic cells have shown that cell replacement grafts in PD are susceptible to Lewy body formation suggesting hostâtoâgraft transfer of αâsynuclein pathology. Here, we have used CRISPR/Cas9n technology to delete the endogenous SNCA gene, encoding for αâsynuclein, in a clinicalâgrade hESC line to generate SNCA+/â and SNCAâ/â cell lines. These hESC lines were first differentiated into mDA neurons, and then challenged with recombinant αâsynuclein preformed fibrils (PFFs) to seed the formation for Lewyâlike pathology as measured by phosphorylation of serineâ129 of αâsynuclein (pS129âαSyn). Wildâtype neurons were fully susceptible to the formation of protein aggregates positive for pS129âαSyn, while SNCA+/â and SNCAâ/â neurons exhibited significant resistance to the formation of this pathological mark. This work demonstrates that reducing or completely removing SNCA alleles by CRISPR/Cas9nâmediated gene editing confers a measure of resistance to Lewy pathology
Viscous regularization and r-adaptive remeshing for finite element analysis of lipid membrane mechanics
As two-dimensional fluid shells, lipid bilayer membranes resist bending and
stretching but are unable to sustain shear stresses. This property gives
membranes the ability to adopt dramatic shape changes. In this paper, a finite
element model is developed to study static equilibrium mechanics of membranes.
In particular, a viscous regularization method is proposed to stabilize
tangential mesh deformations and improve the convergence rate of nonlinear
solvers. The Augmented Lagrangian method is used to enforce global constraints
on area and volume during membrane deformations. As a validation of the method,
equilibrium shapes for a shape-phase diagram of lipid bilayer vesicle are
calculated. These numerical techniques are also shown to be useful for
simulations of three-dimensional large-deformation problems: the formation of
tethers (long tube-like exetensions); and Ginzburg-Landau phase separation of a
two-lipid-component vesicle. To deal with the large mesh distortions of the
two-phase model, modification of vicous regularization is explored to achieve
r-adaptive mesh optimization
Dynamic Fluctuation Phenomena in Double Membrane Films
Dynamics of double membrane films is investigated in the long-wavelength
limit including the overdamped squeezing mode. We demonstrate that thermal
fluctuations essentially modify the character of the mode due to its nonlinear
coupling to the transversal shear hydrodynamic mode. The corresponding Green
function acquires as a function of the frequency a cut along the imaginary
semi-axis. Fluctuations lead to increasing the attenuation of the squeezing
mode it becomes larger than the `bare' value.Comment: 7 pages, Revte
Generalization Strategies in Finding the <i>n</i>th Term Rule for Simple Quadratic Sequences
In this study, we identify ways in which a sample of 18 graduates with mathematics-related first degrees found the nth term for quadratic sequences from the first values of a sequence of data, presented on a computer screen in various formats: tabular, scattered data pairs and sequential. Participantsâ approaches to identifying the nth term were recorded with eye-tracking technology. Our aims are to identify their strategies and to explore whether and how format influences these strategies. Qualitative analysis of eye-tracking data offers several strategies: Sequence of Differences, Building a Relationship, Known Formula, Linear Recursive and Initial Conjecture. Sequence of Differences was the most common strategy, but Building a Relationship was more likely to lead to the right formula. Building from Square and Factor Search were the most successful methods of Building a Relationship. Findings about the influence of format on the range of strategies were inconclusive but analysis indicated sporadic evidence of possible influences
An in vitro model of early anteroposterior organization during human development.
The body plan of the mammalian embryo is shaped through the process of gastrulation, an early developmental event that transforms an isotropic group of cells into an ensemble of tissues that is ordered with reference to three orthogonal axes1. Although model organisms have provided much insight into this process, we know very little about gastrulation in humans, owing to the difficulty of obtaining embryos at such early stages of development and the ethical and technical restrictions that limit the feasibility of observing gastrulation ex vivo2. Here we show that human embryonic stem cells can be used to generate gastruloids-three-dimensional multicellular aggregates that differentiate to form derivatives of the three germ layers organized spatiotemporally, without additional extra-embryonic tissues. Human gastruloids undergo elongation along an anteroposterior axis, and we use spatial transcriptomics to show that they exhibit patterned gene expression. This includes a signature of somitogenesis that suggests that 72-h human gastruloids show some features of Carnegie-stage-9 embryos3. Our study represents an experimentally tractable model system to reveal and examine human-specific regulatory processes that occur during axial organization in early development
Heavy quarkonium: progress, puzzles, and opportunities
A golden age for heavy quarkonium physics dawned a decade ago, initiated by
the confluence of exciting advances in quantum chromodynamics (QCD) and an
explosion of related experimental activity. The early years of this period were
chronicled in the Quarkonium Working Group (QWG) CERN Yellow Report (YR) in
2004, which presented a comprehensive review of the status of the field at that
time and provided specific recommendations for further progress. However, the
broad spectrum of subsequent breakthroughs, surprises, and continuing puzzles
could only be partially anticipated. Since the release of the YR, the BESII
program concluded only to give birth to BESIII; the -factories and CLEO-c
flourished; quarkonium production and polarization measurements at HERA and the
Tevatron matured; and heavy-ion collisions at RHIC have opened a window on the
deconfinement regime. All these experiments leave legacies of quality,
precision, and unsolved mysteries for quarkonium physics, and therefore beg for
continuing investigations. The plethora of newly-found quarkonium-like states
unleashed a flood of theoretical investigations into new forms of matter such
as quark-gluon hybrids, mesonic molecules, and tetraquarks. Measurements of the
spectroscopy, decays, production, and in-medium behavior of c\bar{c}, b\bar{b},
and b\bar{c} bound states have been shown to validate some theoretical
approaches to QCD and highlight lack of quantitative success for others. The
intriguing details of quarkonium suppression in heavy-ion collisions that have
emerged from RHIC have elevated the importance of separating hot- and
cold-nuclear-matter effects in quark-gluon plasma studies. This review
systematically addresses all these matters and concludes by prioritizing
directions for ongoing and future efforts.Comment: 182 pages, 112 figures. Editors: N. Brambilla, S. Eidelman, B. K.
Heltsley, R. Vogt. Section Coordinators: G. T. Bodwin, E. Eichten, A. D.
Frawley, A. B. Meyer, R. E. Mitchell, V. Papadimitriou, P. Petreczky, A. A.
Petrov, P. Robbe, A. Vair
Oxaliplatin/capecitabine or carboplatin/paclitaxel-based preoperative chemoradiation for resectable oesophageal adenocarcinoma (NeoSCOPE): Long-term results of a randomised controlled trial
Aim
This is the first randomised study to evaluate toxicity and survival outcomes of two neoadjuvant chemoradiotherapy (CRT) regimens for patients with localised oesophageal adenocarcinoma (OAC) or gastro-oesophageal junction (GOJ) adenocarcinoma. The initial results showed comparable toxicity between regimens and pathological complete response (pCR) rate favouring CarPacRT. Herein, we report survival, progression patterns, and long-term toxicity after a median follow-up of 40.7 months.
Methods
NeoSCOPE was an open-label, UK multicentre, randomised, phase II trial. Eighty-five patients with resectable OAC or GOJ adenocarcinoma, â„cT3 and/or â„cN1 (TNM v7), suitable for neoadjuvant CRT, were recruited between October 2013 and February 2015.
Patients were randomised to OxCapRT (oxaliplatin 85 mg/m2 on Days 1, 15, and 29; capecitabine 625 mg/m2 orally twice daily on days of radiotherapy [RT]) or CarPacRT (carboplatin AUC2; paclitaxel 50 mg/m2 on Days 1, 8, 15, 22, and 29). RT dose was 45 Gy/25 fractions/5 weeks. Both arms received induction chemotherapy (two cycles oxaliplatin 130 mg/m2 on Day 1, capecitabine 625 mg/m2 orally twice daily on Days 1â21) before CRT. Surgery was performed 6â8 weeks after CRT.
The primary end-point was pCR. Secondary end-points were toxicity, progression-free survival (PFS), overall survival (OS), and patterns of progression.
Results
Eighty-five patients were recruited from 17 UK centres. The median OS was 41.7 months (95% confidence interval [CI] 19.6 to not reached) in the OxCapRT arm and was not reached in the CarPacRT arm (multivariable hazard ratio [HR] = 0.48, 95% CIs: 0.24â0.95, P = 0.035). The median PFS was 32.6 months (95% CIs: 17.1 to not reached) in the OxCapRT arm and was not reached in the CarPacRT arm (multivariable HR = 0.54, 95% CIs: 0.29â1.01, P = 0.053). In both arms, the distant progression was twice as common as locoregional progression.
Conclusions
OS and PFS favoured neoadjuvant CarPacRT over OxCapRT. Distant was more common than locoregional progression; therefore, priority should be given to optimising the systemic treatment component.
Clinical trial information
EudraCT Number: 2012-000640-10; ClinicalTrials.gov: NCT01843829
Potential for pancreatic maturation of differentiating human embryonic stem cells is sensitive to the specific pathway of definitive endoderm commitment
This study provides a detailed experimental and mathematical analysis of the impact of the initial pathway of definitive endoderm (DE) induction on later stages of pancreatic maturation. Human embryonic stem cells (hESCs) were induced to insulin-producing cells following a directed-differentiation approach. DE was induced following four alternative pathway modulations. DE derivatives obtained from these alternate pathways were subjected to pancreatic progenitor (PP) induction and maturation and analyzed at each stage. Results indicate that late stage maturation is influenced by the initial pathway of DE commitment. Detailed quantitative analysis revealed WNT3A and FGF2 induced DE cells showed highest expression of insulin, are closely aligned in gene expression patterning and have a closer resemblance to pancreatic organogenesis. Conversely, BMP4 at DE induction gave most divergent differentiation dynamics with lowest insulin upregulation, but highest glucagon upregulation. Additionally, we have concluded that early analysis of PP markers is indicative of its potential for pancreatic maturation. © 2014 Jaramillo et al
ERK2 Suppresses Self-Renewal Capacity of Embryonic Stem Cells, but Is Not Required for Multi-Lineage Commitment
Activation of the FGF-ERK pathway is necessary for naĂŻve mouse embryonic stem (ES) cells to exit self-renewal and commit to early differentiated lineages. Here we show that genetic ablation of Erk2, the predominant ERK isozyme expressed in ES cells, results in hyper-phosphorylation of ERK1, but an overall decrease in total ERK activity as judged by substrate phosphorylation and immediate-early gene (IEG) induction. Normal induction of this subset of canonical ERK targets, as well as p90RSK phosphorylation, was rescued by transgenic expression of either ERK1 or ERK2 indicating a degree of functional redundancy. In contrast to previously published work, Erk2-null ES cells exhibited no detectable defect in lineage specification to any of the three germ layers when induced to differentiate in either embryoid bodies or in defined neural induction conditions. However, under self-renewing conditions Erk2-null ES cells express increased levels of the pluripotency-associated transcripts, Nanog and Tbx3, a decrease in Nanog-GFP heterogeneity, and exhibit enhanced self-renewal in colony forming assays. Transgenic add-back of ERK2 is capable of restoring normal pluripotent gene expression and self-renewal capacity. We show that ERK2 contributes to the destabilization of ES cell self-renewal by reducing expression of pluripotency genes, such as Nanog, but is not specifically required for the early stages of germ layer specification
Early In Vitro Differentiation of Mouse Definitive Endoderm Is Not Correlated with Progressive Maturation of Nuclear DNA Methylation Patterns
The genome organization in pluripotent cells undergoing the first steps of differentiation is highly relevant to the reprogramming process in differentiation. Considering this fact, chromatin texture patterns that identify cells at the very early stage of lineage commitment could serve as valuable tools in the selection of optimal cell phenotypes for regenerative medicine applications. Here we report on the first-time use of high-resolution three-dimensional fluorescence imaging and comprehensive topological cell-by-cell analyses with a novel image-cytometrical approach towards the identification of in situ global nuclear DNA methylation patterns in early endodermal differentiation of mouse ES cells (up to day 6), and the correlations of these patterns with a set of putative markers for pluripotency and endodermal commitment, and the epithelial and mesenchymal character of cells. Utilizing this in vitro cell system as a model for assessing the relationship between differentiation and nuclear DNA methylation patterns, we found that differentiating cell populations display an increasing number of cells with a gain in DNA methylation load: first within their euchromatin, then extending into heterochromatic areas of the nucleus, which also results in significant changes of methylcytosine/global DNA codistribution patterns. We were also able to co-visualize and quantify the concomitant stochastic marker expression on a per-cell basis, for which we did not measure any correlation to methylcytosine loads or distribution patterns. We observe that the progression of global DNA methylation is not correlated with the standard transcription factors associated with endodermal development. Further studies are needed to determine whether the progression of global methylation could represent a useful signature of cellular differentiation. This concept of tracking epigenetic progression may prove useful in the selection of cell phenotypes for future regenerative medicine applications
- âŠ