Suv4-20h Histone Methyltransferases Promote Neuroectodermal Differentiation by Silencing the Pluripotency-Associated Oct-25 Gene

Post-translational modifications (PTMs) of histones exert fundamental roles in regulating gene expression. During development, groups of PTMs are constrained by unknown mechanisms into combinatorial patterns, which facilitate transitions from uncommitted embryonic cells into differentiated somatic cell lineages. Repressive histone modifications such as H3K9me3 or H3K27me3 have been investigated in detail, but the role of H4K20me3 in development is currently unknown. Here we show that Xenopus laevis Suv4-20h1 and h2 histone methyltransferases (HMTases) are essential for induction and differentiation of the neuroectoderm. Morpholino-mediated knockdown of the two HMTases leads to a selective and specific downregulation of genes controlling neural induction, thereby effectively blocking differentiation of the neuroectoderm. Global transcriptome analysis supports the notion that these effects arise from the transcriptional deregulation of specific genes rather than widespread, pleiotropic effects. Interestingly, morphant embryos fail to repress the Oct4-related Xenopus gene Oct-25. We validate Oct-25 as a direct target of xSu4-20h enzyme mediated gene repression, showing by chromatin immunoprecipitaton that it is decorated with the H4K20me3 mark downstream of the promoter in normal, but not in double-morphant, embryos. Since knockdown of Oct-25 protein significantly rescues the neural differentiation defect in xSuv4-20h double-morphant embryos, we conclude that the epistatic relationship between Suv4-20h enzymes and Oct-25 controls the transit from pluripotent to differentiation-competent neural cells. Consistent with these results in Xenopus, murine Suv4-20h1/h2 double-knockout embryonic stem (DKO ES) cells exhibit increased Oct4 protein levels before and during EB formation, and reveal a compromised and biased capacity for in vitro differentiation, when compared to normal ES cells. Together, these results suggest a regulatory mechanism, conserved between amphibians and mammals, in which H4K20me3-dependent restriction of specific POU-V genes directs cell fate decisions, when embryonic cells exit the pluripotent state

The Chemical Evolution of the La0.6Sr0.4CoO3−δ Surface Under SOFC Operating Conditions and Its Implications for Electrochemical Oxygen Exchange Activity

© The Author(s) 2018Owing to its extraordinary high activity for catalysing the oxygen exchange reaction, strontium doped LaCoO3 (LSC) is one of the most promising materials for solid oxide fuel cell (SOFC) cathodes. However, under SOFC operating conditions this material suffers from performance degradation. This loss of electrochemical activity has been extensively studied in the past and an accumulation of strontium at the LSC surface has been shown to be responsible for most of the degradation effects. The present study sheds further light onto LSC surface changes also occurring under SOFC operating conditions. In-situ near ambient pressure X-ray photoelectron spectroscopy measurements were conducted at temperatures between 400 and 790 °C. Simultaneously, electrochemical impedance measurements were performed to characterise the catalytic activity of the LSC electrode surface for O2 reduction. This combination allowed a correlation of the loss in electro-catalytic activity with the appearance of an additional La-containing Sr-oxide species at the LSC surface. This additional Sr-oxide species preferentially covers electrochemically active Co sites at the surface, and thus very effectively decreases the oxygen exchange performance of LSC. Formation of precipitates, in contrast, was found to play a less important role for the electrochemical degradation of LSC.Fonds zur Förderung der wissenschaftlichen Forschung (FWF)212921411

All-arthroscopic versus mini-open repair of small or moderate-sized rotator cuff tears: A protocol for a randomized trial [NCT00128076]

BACKGROUND: Rotator cuff tears are the most common source of shoulder pain and disability. Only poor quality studies have compared mini-open to arthroscopic repair, leaving surgeons with inadequate evidence to support optimal, minimally-invasive repair. METHODS/DESIGN: This randomized, multi-centre, national trial will determine whether an arthroscopic or mini-open repair provides better quality of life for patients with small or moderate-sized rotator cuff tears. A national consensus meeting of investigators in the Joint Orthopaedic Initiative for National Trials of the Shoulder (JOINTS Canada) identified this question as the top priority for shoulder surgeons across Canada. The primary outcome measure is a valid quality-of-life scale (Western Ontario Rotator Cuff (WORC)) that addresses 5 domains of health affected by rotator cuff disease. Secondary outcomes will assess rotator cuff functionality (ROM, strength, Constant score), secondary dimensions of health (general health status (SF-12) and work limitations), and repair integrity (MRI). Outcomes are measured at baseline, at 6 weeks, 3, 6, 12, and 24 months post-operatively by blinded research assistants and musculoskeletal radiologists. Patients (n = 250) with small or medium-sized cuff tears identified by clinical examination and MRI who meet eligibility criteria will be recruited. This sample size will provide 80% power to statistically detect a clinically important difference of 20% in WORC scores between procedures after controlling for baseline WORC score (α = 0.05). A central methods centre will manage randomization, data management, and monitoring under supervision of experienced epidemiologists. Surgeons will participate in either conventional or expertise-based designs according to defined criteria to avoid biases from differential surgeon expertise. Mini-open or all-arthroscopic repair procedures will be performed according to a standardized protocol. Central Adjudication (of cases), Trial Oversight and Safety Committees will monitor trial conduct. We will use an analysis of covariance (ANCOVA), where the baseline WORC score is used as a covariate, to compare the quality of life (WORC score) at 2 years post-operatively. As a secondary analysis, we will conduct the same statistical test but will include age and tear size as covariates with the baseline score. Enrollment will require 2 years and follow-up an additional 2 years. The trial will commence when funding is in place. DISCUSSION: These results will have immediate impact on the practice behaviors of practicing surgeons and surgical trainees at JOINTS centres across Canada. JOINTS Canada is actively engaged in knowledge exchange and will publish and present findings internationally to facilitate wider application. This trial will establish definitive evidence on this question at an international level

Assessment of ePrescription quality: an observational study at three mail-order pharmacies

<p>Abstract</p> <p>Background</p> <p>The introduction of electronic transfer of prescriptions (ETP) or ePrescriptions in ambulatory health care has been suggested to have a positive impact on the prescribing and dispensing processes. Thereby, implying that ePrescribing can improve safety, quality, efficiency, and cost-effectiveness. In December 2007, 68% of all new prescriptions were transferred electronically in Sweden. The aim of the present study was to assess the quality of ePrescriptions by comparing the proportions of ePrescriptions and non-electronic prescriptions necessitating a clarification contact (correction, completion or change) with the prescriber at the time of dispensing.</p> <p>Methods</p> <p>A direct observational study was performed at three Swedish mail-order pharmacies which were known to dispense a large proportion of ePrescriptions (38–75%). Data were gathered on all ePrescriptions dispensed at these pharmacies over a three week period in February 2006. All clarification contacts with prescribers were included in the study and were classified and assessed in comparison with all drug prescriptions dispensed at the same pharmacies over the specified period.</p> <p>Results</p> <p>Of the 31225 prescriptions dispensed during the study period, clarification contacts were made for 2.0% (147/7532) of new ePrescriptions and 1.2% (79/6833) of new non-electronic prescriptions. This represented a relative risk (RR) of 1.7 (95% CI 1.3–2.2) for new ePrescriptions compared to new non-electronic prescriptions. The increased RR was mainly due to 'Dosage and directions for use', which had an RR of 7.6 (95% CI 2.8–20.4) when compared to other clarification contacts. In all, 89.5% of the suggested pharmacist interventions were accepted by the prescriber, 77.7% (192/247) as suggested and an additional 11.7% (29/247) after a modification during contact with the prescriber.</p> <p>Conclusion</p> <p>The increased proportion of prescriptions necessitating a clarification contact for new ePrescriptions compared to new non-electronic prescriptions indicates the need for an increased focus on quality aspects in ePrescribing deployment. ETP technology should be developed towards a two-way communication between the prescriber and the pharmacist with automated checks of missing, inaccurate, or ambiguous information. This would enhance safety and quality for the patient and also improve efficiency and cost-effectiveness within the health care system.</p

Influence of surface atomic structure demonstrated on oxygen incorporation mechanism at a model perovskite oxide

Perovskite oxide surfaces catalyze oxygen exchange reactions that are crucial for fuel cells, electrolyzers, and thermochemical fuel synthesis. Here, by bridging the gap between surface analysis with atomic resolution and oxygen exchange kinetics measurements, we demonstrate how the exact surface atomic structure can determine the reactivity for oxygen exchange reactions on a model perovskite oxide. Two precisely controlled surface reconstructions with (4 × 1) and (2 × 5) symmetry on 0.5 wt.% Nb-doped SrTiO3(110) were subjected to isotopically labeled oxygen exchange at 450 °C. The oxygen incorporation rate is three times higher on the (4 × 1) surface phase compared to the (2 × 5). Common models of surface reactivity based on the availability of oxygen vacancies or on the ease of electron transfer cannot account for this difference. We propose a structure-driven oxygen exchange mechanism, relying on the flexibility of the surface coordination polyhedra that transform upon dissociation of oxygen molecules.Austrian Science Fund (SFB “ Functional Oxide Surfaces and Interfaces ” - FOXSI, Project F 45)European Research Council Advanced Grant (“OxideSurfaces” (Project ERC-2011-ADG_20110209))National Science Foundation (U.S.). Division of Materials Research (CAREER Award Grant No. 1055583

Expression profiling with RNA from formalin-fixed, paraffin-embedded material

<p>Abstract</p> <p>Background</p> <p>Molecular characterization of breast and other cancers by gene expression profiling has corroborated existing classifications and revealed novel subtypes. Most profiling studies are based on fresh frozen (FF) tumor material which is available only for a limited number of samples while thousands of tumor samples exist as formalin-fixed, paraffin-embedded (FFPE) blocks. Unfortunately, RNA derived of FFPE material is fragmented and chemically modified impairing expression measurements by standard procedures. Robust protocols for isolation of RNA from FFPE material suitable for stable and reproducible measurement of gene expression (e.g. by quantitative reverse transcriptase PCR, QPCR) remain a major challenge.</p> <p>Results</p> <p>We present a simple procedure for RNA isolation from FFPE material of diagnostic samples. The RNA is suitable for expression measurement by QPCR when used in combination with an optimized cDNA synthesis protocol and TaqMan assays specific for short amplicons. The FFPE derived RNA was compared to intact RNA isolated from the same tumors. Preliminary scores were computed from genes related to the ER response, HER2 signaling and proliferation. Correlation coefficients between intact and partially fragmented RNA from FFPE material were 0.83 to 0.97.</p> <p>Conclusion</p> <p>We developed a simple and robust method for isolating RNA from FFPE material. The RNA can be used for gene expression profiling. Expression measurements from several genes can be combined to robust scores representing the hormonal or the proliferation status of the tumor.</p

Anchor Side Chains of Short Peptide Fragments Trigger Ligand-Exchange of Class II MHC Molecules

Class II MHC molecules display peptides on the cell surface for the surveillance by CD4+ T cells. To ensure that these ligands accurately reflect the content of the intracellular MHC loading compartment, a complex processing pathway has evolved that delivers only stable peptide/MHC complexes to the surface. As additional safeguard, MHC molecules quickly acquire a ‘non-receptive’ state once they have lost their ligand. Here we show now that amino acid side chains of short peptides can bypass these safety mechanisms by triggering the reversible ligand-exchange. The catalytic activity of dipeptides such as Tyr-Arg was stereo-specific and could be enhanced by modifications addressing the conserved H-bond network near the P1 pocket of the MHC molecule. It affected both antigen-loading and ligand-release and strictly correlated with reported anchor preferences of P1, the specific target site for the catalytic side chain of the dipeptide. The effect was evident also in CD4+ T cell assays, where the allele-selective influence of the dipeptides translated into increased sensitivities of the antigen-specific immune response. Molecular dynamic calculations support the hypothesis that occupation of P1 prevents the ‘closure’ of the empty peptide binding site into the non-receptive state. During antigen-processing and -presentation P1 may therefore function as important “sensor” for peptide-load. While it regulates maturation and trafficking of the complex, on the cell surface, short protein fragments present in blood or lymph could utilize this mechanism to alter the ligand composition on antigen presenting cells in a catalytic way

Clinical relevance of DNA microarray analyses using archival formalin-fixed paraffin-embedded breast cancer specimens

Abstract Background The ability of gene profiling to predict treatment response and prognosis in breast cancers has been demonstrated in many studies using DNA microarray analyses on RNA from fresh frozen tumor specimens. In certain clinical and research situations, performing such analyses on archival formalin fixed paraffin-embedded (FFPE) surgical specimens would be advantageous as large libraries of such specimens with long-term follow-up data are widely available. However, FFPE tissue processing can cause fragmentation and chemical modifications of the RNA. A number of recent technical advances have been reported to overcome these issues. Our current study evaluates whether or not the technology is ready for clinical applications. Methods A modified RNA extraction method and a recent DNA microarray technique, cDNA-mediated annealing, selection, extension and ligation (DASL, Illumina Inc) were evaluated. The gene profiles generated from FFPE specimens were compared to those obtained from paired fresh fine needle aspiration biopsies (FNAB) of 25 breast cancers of different clinical subtypes (based on ER and Her2/neu status). Selected RNA levels were validated using RT-qPCR, and two public databases were used to demonstrate the prognostic significance of the gene profiles generated from FFPE specimens. Results Compared to FNAB, RNA isolated from FFPE samples was relatively more degraded, nonetheless, over 80% of the RNA samples were deemed suitable for subsequent DASL assay. Despite a higher noise level, a set of genes from FFPE specimens correlated very well with the gene profiles obtained from FNAB, and could differentiate breast cancer subtypes. Expression levels of these genes were validated using RT-qPCR. Finally, for the first time we correlated gene expression profiles from FFPE samples to survival using two independent microarray databases. Specifically, over-expression of ANLN and KIF2C, and under-expression of MAPT strongly correlated with poor outcomes in breast cancer patients. Conclusion We demonstrated that FFPE specimens retained important prognostic information that could be identified using a recent gene profiling technology. Our study supports the use of FFPE specimens for the development and refinement of prognostic gene signatures for breast cancer. Clinical applications of such prognostic gene profiles await future large-scale validation studies

Characterization of Structural Features Controlling the Receptiveness of Empty Class II MHC Molecules

MHC class II molecules (MHC II) play a pivotal role in the cell-surface presentation of antigens for surveillance by T cells. Antigen loading takes place inside the cell in endosomal compartments and loss of the peptide ligand rapidly leads to the formation of a non-receptive state of the MHC molecule. Non-receptiveness hinders the efficient loading of new antigens onto the empty MHC II. However, the mechanisms driving the formation of the peptide inaccessible state are not well understood. Here, a combined approach of experimental site-directed mutagenesis and computational modeling is used to reveal structural features underlying “non-receptiveness.” Molecular dynamics simulations of the human MHC II HLA-DR1 suggest a straightening of the α-helix of the β1 domain during the transition from the open to the non-receptive state. The movement is mostly confined to a hinge region conserved in all known MHC molecules. This shift causes a narrowing of the two helices flanking the binding site and results in a closure, which is further stabilized by the formation of a critical hydrogen bond between residues αQ9 and βN82. Mutagenesis experiments confirmed that replacement of either one of the two residues by alanine renders the protein highly susceptible. Notably, loading enhancement was also observed when the mutated MHC II molecules were expressed on the surface of fibroblast cells. Altogether, structural features underlying the non-receptive state of empty HLA-DR1 identified by theoretical means and experiments revealed highly conserved residues critically involved in the receptiveness of MHC II. The atomic details of rearrangements of the peptide-binding groove upon peptide loss provide insight into structure and dynamics of empty MHC II molecules and may foster rational approaches to interfere with non-receptiveness. Manipulation of peptide loading efficiency for improved peptide vaccination strategies could be one of the applications profiting from the structural knowledge provided by this study

Mechanisms of local immunosuppression in cutaneous melanoma

Cutaneous melanoma is highly immunogenic, yet primary melanomas and metastases develop successfully in otherwise immunocompetent patients. To investigate the local immunosuppressive microenvironment, we examined the presence of suppressor T lymphocytes and tolerising dendritic cells (DCs), the expression of immunosuppressive cytokines (IL-10, TGFβ1 and TGFβ2) and the enzyme indoleamine 2,3-dioxygenase (IDO) using qRT–PCR and immunohistochemistry in primary skin melanomas, negative and positive sentinel lymph nodes (SLN), and lymph nodes with advanced metastases. Our results indicate that tolerogenic DCs and suppressor T lymphocytes are present in melanoma at all stages of disease progression. They express transforming growth factor β receptor 1 (TGFβR1), and are therefore susceptible to TGFβ1 and TGFβ2 specifically expressed by primary melanoma. We found that expression of IDO and interleukin 10 (IL-10) increased with melanoma progression, with the highest concentration in positive SLN. We suggest that negative SLN contain immunosuppressive cells and cytokines, due to preconditioning by tolerogenic DCs migrating from the primary melanoma site to the SLN. In primary melanoma, TGFβ2 is likely to render peripheral DCs tolerogenic, while in lymph nodes IDO and TGFβ1 may have a major effect. This mechanism of tumour-associated immunosuppression may inhibit the immune response to the tumour and may explain the discrepancy between the induction of systemic immunity by anti-melanoma vaccines and their poor performance in the clinic