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

Applying refinement to the use of mice and rats in rheumatoid arthritis research

Rheumatoid arthritis (RA) is a painful, chronic disorder and there is currently an unmet need for effective therapies that will benefit a wide range of patients. The research and development process for therapies and treatments currently involves in vivo studies, which have the potential to cause discomfort, pain or distress. This Working Group report focuses on identifying causes of suffering within commonly used mouse and rat ‘models’ of RA, describing practical refinements to help reduce suffering and improve welfare without compromising the scientific objectives. The report also discusses other, relevant topics including identifying and minimising sources of variation within in vivo RA studies, the potential to provide pain relief including analgesia, welfare assessment, humane endpoints, reporting standards and the potential to replace animals in RA research

Increasing the Analytical Sensitivity by Oligonucleotides Modified with Para- and Ortho-Twisted Intercalating Nucleic Acids – TINA

The sensitivity and specificity of clinical diagnostic assays using DNA hybridization techniques are limited by the dissociation of double-stranded DNA (dsDNA) antiparallel duplex helices. This situation can be improved by addition of DNA stabilizing molecules such as nucleic acid intercalators. Here, we report the synthesis of a novel ortho-Twisted Intercalating Nucleic Acid (TINA) amidite utilizing the phosphoramidite approach, and examine the stabilizing effect of ortho- and para-TINA molecules in antiparallel DNA duplex formation. In a thermal stability assay, ortho- and para-TINA molecules increased the melting point (Tm) of Watson-Crick based antiparallel DNA duplexes. The increase in Tm was greatest when the intercalators were placed at the 5′ and 3′ termini (preferable) or, if placed internally, for each half or whole helix turn. Terminally positioned TINA molecules improved analytical sensitivity in a DNA hybridization capture assay targeting the Escherichia coli rrs gene. The corresponding sequence from the Pseudomonas aeruginosa rrs gene was used as cross-reactivity control. At 150 mM ionic strength, analytical sensitivity was improved 27-fold by addition of ortho-TINA molecules and 7-fold by addition of para-TINA molecules (versus the unmodified DNA oligonucleotide), with a 4-fold increase retained at 1 M ionic strength. Both intercalators sustained the discrimination of mismatches in the dsDNA (indicated by ΔTm), unless placed directly adjacent to the mismatch – in which case they partly concealed ΔTm (most pronounced for para-TINA molecules). We anticipate that the presented rules for placement of TINA molecules will be broadly applicable in hybridization capture assays and target amplification systems

Whole body vibration compared to conventional physiotherapy in patients with gonarthrosis: a protocol for a randomized, controlled study

<p>Abstract</p> <p>Background</p> <p>Osteoarthritis (OA) is the most common degenerative arthropathy. Load-bearing joints such as knee and hip are more often affected than spine or hands. The prevalence of gonarthrosis is generally higher than that of coxarthrosis.</p> <p>Because no cure for OA exists, the main emphasis of therapy is analgesic treatment through either mobility or medication. Non-pharmacologic treatment is the first step, followed by the addition of analgesic medication, and ultimately by surgery.</p> <p>The goal of non-pharmacologic and non-invasive therapy is to improve neuromuscular function, which in turn both prevents formation of and delays progression of OA. A modification of conventional physiotherapy, whole body vibration has been successfully employed for several years. Since its introduction, this therapy is in wide use at our facility not only for gonarthrosis, but also coxarthrosis and other diseases leading to muscular imbalance.</p> <p>Methods/Design</p> <p>This study is a randomized, therapy-controlled trial in a primary care setting at a university hospital. Patients presenting to our outpatient clinic with initial symptoms of gonarthrosis will be assessed against inclusion and exclusion criteria. After patient consent, 6 weeks of treatment will ensue. During the six weeks of treatment, patients will receive one of two treatments, conventional physiotherapy or whole-body-vibration exercises of one hour three times a week. Follow-up examinations will be performed immediately after treatment and after another 6 and 20 weeks, for a total study duration of 6 months. 20 patients will be included in each therapy group.</p> <p>Outcome measurements will include objective analysis of motion and ambulation as well as examinations of balance and isokinetic force. The Western Ontario and McMaster Universities Arthritis Index and SF-12 scores, the patients' overall status, and clinical examinations of the affected joint will be carried out.</p> <p>Discussion</p> <p>As new physiotherapy techniques develop for the treatment of OA, it is important to investigate the effectiveness of competing strategies. With this study, not only patient-based scores, but also objective assessments will be used to quantify patient-derived benefits of therapy.</p> <p>Trial registration</p> <p>Deutsches Register Klinischer Studien (DRKS) DRKS00000415</p> <p>Clinicaltrials.gov NCT01037972</p> <p>EudraCT 2009-017617-29</p

Consensus on circulatory shock and hemodynamic monitoring. Task force of the European Society of Intensive Care Medicine.

OBJECTIVE: Circulatory shock is a life-threatening syndrome resulting in multiorgan failure and a high mortality rate. The aim of this consensus is to provide support to the bedside clinician regarding the diagnosis, management and monitoring of shock. METHODS: The European Society of Intensive Care Medicine invited 12 experts to form a Task Force to update a previous consensus (Antonelli et al.: Intensive Care Med 33:575-590, 2007). The same five questions addressed in the earlier consensus were used as the outline for the literature search and review, with the aim of the Task Force to produce statements based on the available literature and evidence. These questions were: (1) What are the epidemiologic and pathophysiologic features of shock in the intensive care unit ? (2) Should we monitor preload and fluid responsiveness in shock ? (3) How and when should we monitor stroke volume or cardiac output in shock ? (4) What markers of the regional and microcirculation can be monitored, and how can cellular function be assessed in shock ? (5) What is the evidence for using hemodynamic monitoring to direct therapy in shock ? Four types of statements were used: definition, recommendation, best practice and statement of fact. RESULTS: Forty-four statements were made. The main new statements include: (1) statements on individualizing blood pressure targets; (2) statements on the assessment and prediction of fluid responsiveness; (3) statements on the use of echocardiography and hemodynamic monitoring. CONCLUSIONS: This consensus provides 44 statements that can be used at the bedside to diagnose, treat and monitor patients with shock

HOXB4 Gene Expression Is Regulated by CDX2 in Intestinal Epithelial Cells

The mammalian Caudal-related homeobox transcription factor 2 (CDX2) plays a key role in the homeobox regulatory network and is essential in regulating the expression of several homeobox (HOX) genes during embryonic development, particularly in the gut. Genome-wide CDX2 chromatin immunoprecipitation analysis and expression data from Caco2 cells also suggests a role for CDX2 in the regulation of HOXB4 gene expression in the intestinal epithelium. Thus, the aim of this study was to investigate whether HOXB4 gene expression is regulated by CDX2 in the intestinal epithelium. We demonstrated binding of CDX2 to four different CDX2 binding sites in an enhancer region located upstream of the HOXB4 transcription start site. Mutations in the CDX2 binding sites reduced HOXB4 gene activity, and knock down of endogenous CDX2 expression by shRNA reduced HOXB4 gene expression. This is the first report demonstrating the CDX2 regulation of HOXB4 gene expression in the developed intestinal epithelium, indicating a possible role for HOXB4 in intestinal homeostasis

Zelda Binding in the Early Drosophila melanogaster Embryo Marks Regions Subsequently Activated at the Maternal-to-Zygotic Transition

The earliest stages of development in most metazoans are driven by maternally deposited proteins and mRNAs, with widespread transcriptional activation of the zygotic genome occurring hours after fertilization, at a period known as the maternal-to-zygotic transition (MZT). In Drosophila, the MZT is preceded by the transcription of a small number of genes that initiate sex determination, patterning, and other early developmental processes; and the zinc-finger protein Zelda (ZLD) plays a key role in their transcriptional activation. To better understand the mechanisms of ZLD activation and the range of its targets, we used chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) to map regions bound by ZLD before (mitotic cycle 8), during (mitotic cycle 13), and after (late mitotic cycle 14) the MZT. Although only a handful of genes are transcribed prior to mitotic cycle 10, we identified thousands of regions bound by ZLD in cycle 8 embryos, most of which remain bound through mitotic cycle 14. As expected, early ZLD-bound regions include the promoters and enhancers of genes transcribed at this early stage. However, we also observed ZLD bound at cycle 8 to the promoters of roughly a thousand genes whose first transcription does not occur until the MZT and to virtually all of the thousands of known and presumed enhancers bound at cycle 14 by transcription factors that regulate patterned gene activation during the MZT. The association between early ZLD binding and MZT activity is so strong that ZLD binding alone can be used to identify active promoters and regulatory sequences with high specificity and selectivity. This strong early association of ZLD with regions not active until the MZT suggests that ZLD is not only required for the earliest wave of transcription but also plays a major role in activating the genome at the MZT

Optimal stomatal behaviour around the world

© 2015 Macmillan Publishers Limited. All rights reserved. Stomatal conductance (g s) is a key land-surface attribute as it links transpiration, the dominant component of global land evapotranspiration, and photosynthesis, the driving force of the global carbon cycle. Despite the pivotal role of g s in predictions of global water and carbon cycle changes, a global-scale database and an associated globally applicable model of g s that allow predictions of stomatal behaviour are lacking. Here, we present a database of globally distributed g s obtained in the field for a wide range of plant functional types (PFTs) and biomes. We find that stomatal behaviour differs among PFTs according to their marginal carbon cost of water use, as predicted by the theory underpinning the optimal stomatal model and the leaf and wood economics spectrum. We also demonstrate a global relationship with climate. These findings provide a robust theoretical framework for understanding and predicting the behaviour of g s across biomes and across PFTs that can be applied to regional, continental and global-scale modelling of ecosystem productivity, energy balance and ecohydrological processes in a future changing climate

Optimal stomatal behaviour around the world

This is the author accepted manuscript. The final version is available from Springer Nature via the DOI in this recordStomatal conductance (g s) is a key land-surface attribute as it links transpiration, the dominant component of global land evapotranspiration, and photosynthesis, the driving force of the global carbon cycle. Despite the pivotal role of g s in predictions of global water and carbon cycle changes, a global-scale database and an associated globally applicable model of g s that allow predictions of stomatal behaviour are lacking. Here, we present a database of globally distributed g s obtained in the field for a wide range of plant functional types (PFTs) and biomes. We find that stomatal behaviour differs among PFTs according to their marginal carbon cost of water use, as predicted by the theory underpinning the optimal stomatal model and the leaf and wood economics spectrum. We also demonstrate a global relationship with climate. These findings provide a robust theoretical framework for understanding and predicting the behaviour of g s across biomes and across PFTs that can be applied to regional, continental and global-scale modelling of ecosystem productivity, energy balance and ecohydrological processes in a future changing climate.This research was supported by the Australian Research Council (ARC MIA Discovery Project 1433500-2012-14). A.R. was financially supported in part by The Next-Generation Ecosystem Experiments (NGEE-Arctic) project, which is supported by the Office of Biological and Environmental Research in the Department of Energy, Office of Science, and through the United States Department of Energy contract No. DE-AC02-98CH10886 to Brookhaven National Laboratory. M.O.d.B. acknowledges that the Brassica data were obtained within a research project financed by the Belgian Science Policy (OFFQ, contract number SD/AF/02) and coordinated by K. Vandermeiren at the Open-Top Chamber research facilities of CODA-CERVA (Tervuren, Belgium)