Initiating head development in mouse embryos: integrating signalling and transcriptional activity

The generation of an embryonic body plan is the outcome of inductive interactions between the progenitor tissues that underpin their specification, regionalization and morphogenesis. The intercellular signalling activity driving these processes is deployed in a time- and site-specific manner, and the signal strength must be precisely controlled. Receptor and ligand functions are modulated by secreted antagonists to impose a dynamic pattern of globally controlled and locally graded signals onto the tissues of early post-implantation mouse embryo. In response to the WNT, Nodal and Bone Morphogenetic Protein (BMP) signalling cascades, the embryo acquires its body plan, which manifests as differences in the developmental fate of cells located at different positions in the anterior–posterior body axis. The initial formation of the anterior (head) structures in the mouse embryo is critically dependent on the morphogenetic activity emanating from two signalling centres that are juxtaposed with the progenitor tissues of the head. A common property of these centres is that they are the source of antagonistic factors and the hub of transcriptional activities that negatively modulate the function of WNT, Nodal and BMP signalling cascades. These events generate the scaffold of the embryonic head by the early-somite stage of development. Beyond this, additional tissue interactions continue to support the growth, regionalization, differentiation and morphogenesis required for the elaboration of the structure recognizable as the embryonic head

IsoBED: a tool for automatic calculation of biologically equivalent fractionation schedules in radiotherapy using IMRT with a simultaneous integrated boost (SIB) technique

<p>Abstract</p> <p>Background</p> <p>An advantage of the Intensity Modulated Radiotherapy (IMRT) technique is the feasibility to deliver different therapeutic dose levels to PTVs in a single treatment session using the Simultaneous Integrated Boost (SIB) technique. The paper aims to describe an automated tool to calculate the dose to be delivered with the SIB-IMRT technique in different anatomical regions that have the same Biological Equivalent Dose (BED), i.e. IsoBED, compared to the standard fractionation.</p> <p>Methods</p> <p>Based on the Linear Quadratic Model (LQM), we developed software that allows treatment schedules, biologically equivalent to standard fractionations, to be calculated. The main radiobiological parameters from literature are included in a database inside the software, which can be updated according to the clinical experience of each Institute. In particular, the BED to each target volume will be computed based on the alpha/beta ratio, total dose and the dose per fraction (generally 2 Gy for a standard fractionation). Then, after selecting the reference target, i.e. the PTV that controls the fractionation, a new total dose and dose per fraction providing the same isoBED will be calculated for each target volume.</p> <p>Results</p> <p>The IsoBED Software developed allows: 1) the calculation of new IsoBED treatment schedules derived from standard prescriptions and based on LQM, 2) the conversion of the dose-volume histograms (DVHs) for each Target and OAR to a nominal standard dose at 2Gy per fraction in order to be shown together with the DV-constraints from literature, based on the LQM and radiobiological parameters, and 3) the calculation of Tumor Control Probability (TCP) and Normal Tissue Complication Probability (NTCP) curve versus the prescribed dose to the reference target.</p

The epidemiology and transmission of methicillin-resistant Staphylococcus aureus in the community in Singapore: study protocol for a longitudinal household study.

BACKGROUND/AIM: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common multidrug-resistant organisms in healthcare settings worldwide, but little is known about MRSA transmission outside of acute healthcare settings especially in Asia. We describe the methods for a prospective longitudinal study of MRSA prevalence and transmission. METHODS: MRSA-colonized individuals were identified from MRSA admission screening at two tertiary hospitals and recruited together with their household contacts. Participants submitted self-collected nasal, axilla and groin (NAG) swabs by mail for MRSA culture at baseline and monthly thereafter for 6 months. A comparison group of households of MRSA-negative patients provided swab samples at one time point. In a validation sub-study, separate swabs from each site were collected from randomly selected individuals, to compare MRSA detection rates between swab sites, and between samples collected by participants versus those collected by trained research staff. Information on each participant's demographic information, medical status and medical history, past healthcare facilities usage and contacts, and personal interactions with others were collected using a self-administered questionnaire. DISCUSSION/CONCLUSION: Understanding the dynamics of MRSA persistence and transmission in the community is crucial to devising and evaluating successful MRSA control strategies. Close contact with MRSA colonized patients may to be important for MRSA persistence in the community; evidence from this study on the extent of community MRSA could inform the development of household- or community-based interventions to reduce MRSA colonization of close contacts and subsequent re-introduction of MRSA into healthcare settings. Analysis of longitudinal data using whole-genome sequencing will yield further information regarding MRSA transmission within households, with significant implications for MRSA infection control outside acute hospital settings

Genome-Wide Analysis Reveals a Major Role in Cell Fate Maintenance and an Unexpected Role in Endoreduplication for the Drosophila FoxA Gene Fork Head

Transcription factors drive organogenesis, from the initiation of cell fate decisions to the maintenance and implementation of these decisions. The Drosophila embryonic salivary gland provides an excellent platform for unraveling the underlying transcriptional networks of organ development because Drosophila is relatively unencumbered by significant genetic redundancy. The highly conserved FoxA family transcription factors are essential for various aspects of organogenesis in all animals that have been studied. Here, we explore the role of the single Drosophila FoxA protein Fork head (Fkh) in salivary gland organogenesis using two genome-wide strategies. A large-scale in situ hybridization analysis reveals a major role for Fkh in maintaining the salivary gland fate decision and controlling salivary gland physiological activity, in addition to its previously known roles in morphogenesis and survival. The majority of salivary gland genes (59%) are affected by fkh loss, mainly at later stages of salivary gland development. We show that global expression of Fkh cannot drive ectopic salivary gland formation. Thus, unlike the worm FoxA protein PHA-4, Fkh does not function to specify cell fate. In addition, Fkh only indirectly regulates many salivary gland genes, which is also distinct from the role of PHA-4 in organogenesis. Our microarray analyses reveal unexpected roles for Fkh in blocking terminal differentiation and in endoreduplication in the salivary gland and in other Fkh-expressing embryonic tissues. Overall, this study demonstrates an important role for Fkh in determining how an organ preserves its identity throughout development and provides an alternative paradigm for how FoxA proteins function in organogenesis

A novel approach to glaucoma screening and education in Nepal

<p>Abstract</p> <p>Background</p> <p>Glaucoma is a major cause of blindness worldwide and an increasingly significant global health problem. Glaucoma prevention and management efforts have been challenging due to inherent difficulty in developing a simple and cost-effective screening plan, limited access to health care and educational resources, poverty, and inadequate knowledge of the disease, particularly in developing countries. Starting in 2004 the Tilganga Eye Centre in Kathmandu, Nepal has provided targeted glaucoma screening, treatment, and education through a combination of clinical outreach programs and educational activities for patients.</p> <p>Methods</p> <p>A simple, age-based glaucoma screening algorithm was incorporated into three one-day cataract screening clinics. Using this algorithm, patients who were newly diagnosed with glaucoma were referred to TEC, where medication and surgery were provided free of charge through private donor funding. In addition, we describe two ongoing educational programs for increasing glaucoma awareness: an annual Glaucoma Awareness Week (which includes free screening, treatment, and counseling), and a repeating lecture series which generates new counselors.</p> <p>Results</p> <p>From 2004 to 2007 screening at the annual Glaucoma Awareness Week resulted in the diagnosis of 120 individuals with glaucoma, or 7.6% of total registrants. Attendance increased annually with a trend toward an increasing number of returning patients but a decreasing percentage of newly diagnosed patients, though the absolute numbers have remained relatively stable (range 21 to 38). Data from the three one-day screening clinics in 2006 show that approximately 2 to 4% of patients 50 years of age or older per clinic were newly diagnosed with POAG.</p> <p>Conclusion</p> <p>This multi-faceted approach appears to successfully identify individuals with glaucoma and provide treatment to those who would otherwise not be able to afford it. While more data is needed to validate this model, specifically regarding the effectiveness of educational activities, long-term visual outcomes, and medication compliance, it may serve as a useful framework for other developing countries with similarly limited resources.</p

Graded Smad2/3 Activation Is Converted Directly into Levels of Target Gene Expression in Embryonic Stem Cells

The Transforming Growth Factor (TGF) β signalling family includes morphogens, such as Nodal and Activin, with important functions in vertebrate development. The concentration of the morphogen is critical for fate decisions in the responding cells. Smad2 and Smad3 are effectors of the Nodal/Activin branch of TGFβ signalling: they are activated by receptors, enter the nucleus and directly transcribe target genes. However, there have been no studies correlating levels of Smad2/3 activation with expression patterns of endogenous target genes in a developmental context over time. We used mouse Embryonic Stem (ES) cells to create a system whereby levels of activated Smad2/3 can be manipulated by an inducible constitutively active receptor (Alk4*) and an inhibitor (SB-431542) that blocks specifically Smad2/3 activation. The transcriptional responses were analysed by microarrays at different time points during activation and repression. We identified several genes that follow faithfully and reproducibly the Smad2/3 activation profile. Twenty-seven of these were novel and expressed in the early embryo downstream of Smad2/3 signalling. As they responded to Smad2/3 activation in the absence of protein synthesis, they were considered direct. These immediate responsive genes included negative intracellular feedback factors, like SnoN and I-Smad7, which inhibit the transcriptional activity of Smad2/3. However, their activation did not lead to subsequent repression of target genes over time, suggesting that this type of feedback is inefficient in ES cells or it is counteracted by mechanisms such as ubiquitin-mediated degradation by Arkadia. Here we present an ES cell system along with a database containing the expression profile of thousands of genes downstream of Smad2/3 activation patterns, in the presence or absence of protein synthesis. Furthermore, we identify primary target genes that follow proportionately and with high sensitivity changes in Smad2/3 levels over 15–30 hours. The above system and resource provide tools to study morphogen function in development

Culture of Mouse Embryonic Stem Cells with Serum but without Exogenous Growth Factors Is Sufficient to Generate Functional Hepatocyte-Like Cells

Mouse embryonic stem cells (mESC) have been used to study lineage specification in vitro, including towards a hepatocyte-like fate, and such investigations guided lineage differentiation protocols for human (h)ESC. We recently described a four-step protocol to induce hepatocyte-like cells from hESC which also induced hepatocyte-like cell differentiation of mouse induced pluripotent stem cells. As ESC also spontaneously generate hepatocyte-like cells, we here tested whether the growth factors and serum used in this protocol are required to commit mESC and hESC to hepatocyte-like cells. Culture of mESC from two different mouse strains in the absence of serum and growth factors did not induce primitive streak/definitive endoderm genes but induced default differentiation to neuroectoderm on day 6. Although Activin-A and Wnt3 induced primitive streak/definitive endoderm transcripts most robustly in mESC, simple addition of serum also induced these transcripts. Expression of hepatoblast genes occurred earlier when growth factors were used for mESC differentiation. However, further maturation towards functional hepatocyte-like cells was similar in mESC progeny from cultures with serum, irrespective of the addition of growth factors, and irrespective of the mouse strain. This is in contrast to hESC, where growth factors are required for specification towards functional hepatocyte-like cells. Culture of mESC with serum but without growth factors did not induce preferential differentiation towards primitive endoderm or neuroectoderm. Thus, although induction of primitive streak/definitive endoderm specific genes and proteins is more robust when mESC are exposed to a combination of serum and exogenous growth factors, ultimate generation of hepatocyte-like cells from mESC occurs equally well in the presence or absence of exogenous growth factors. The latter is in contrast to what we observed for hESC. These results suggest that differences exist between lineage specific differentiation potential of mESC and hESC, requiring optimization of different protocols for ESC from either species

The placebo effect and its determinants in fibromyalgia: meta-analysis of randomized controlled trials

The aims of this study were to determine whether placebo treatment in randomised controlled trials (RCTs) is effective for fibromyalgia and to identify possible determinants of the magnitude of any such placebo effect. A systematic literature search was undertaken for RCTs in people with fibromyalgia that included a placebo and/or a no-treatment (observation only or waiting list) control group. Placebo effect size (ES) for pain and other outcomes was measured as the improvement of each outcome from baseline divided by the standard deviation of the change from baseline. This effect was compared with changes in the no-treatment control groups. Meta-analysis was undertaken to combine data from different studies. Subgroup analysis was conducted to identify possible determinants of the placebo ES. A total of 3912 studies were identified from the literature search. After scrutiny, 229 trials met the inclusion criteria. Participants who received placebo in the RCTs experienced significantly better improvements in pain, fatigue, sleep quality, physical function, and other main outcomes than those receiving no treatment. The ES of placebo for pain relief was clinically moderate (0.53, 95%CI 0.48 to 0.57). The ES increased with increasing strength of the active treatment, increasing participant age and higher baseline pain severity, but decreased in RCTS with more women and with longer duration of fibromyalgia. In addition, placebo treatment in RCTs is effective in fibromyalgia. A number of factors (expected strength of treatment, age, gender, disease duration) appear to influence the magnitude of the placebo effect in this condition

RNA Interference Mediated Inhibition of Dengue Virus Multiplication and Entry in HepG2 Cells

Background: Dengue virus-host cell interaction initiates when the virus binds to the attachment receptors followed by endocytic internalization of the virus particle. Successful entry into the cell is necessary for infection initiation. Currently, there is no protective vaccine or antiviral treatment for dengue infection. Targeting the viral entry pathway has become an attractive therapeutic strategy to block infection. This study aimed to investigate the effect of silencing the GRP78 and clathrin-mediated endocytosis on dengue virus entry and multiplication into HepG2 cells. Methodology/Principal Findings: HepG2 cells were transfected using specific siRNAs to silence the cellular surface receptor (GRP78) and clathrin-mediated endocytosis pathway. Gene expression analysis showed a marked down-regulation of the targeted genes (87.2%, 90.3%, and 87.8 % for GRP78, CLTC, and DNM2 respectively) in transfected HepG2 cells when measured by RT-qPCR. Intracellular and extracellular viral RNA loads were quantified by RT-qPCR to investigate the effect of silencing the attachment receptor and clathrin-mediated endocytosis on dengue virus entry. Silenced cells showed a significant reduction of intracellular (92.4%) and extracellular viral RNA load (71.4%) compared to non-silenced cells. Flow cytometry analysis showed a marked reduction of infected cells (89.7%) in silenced HepG2 cells compared to non-silenced cells. Furthermore, the ability to generate infectious virions using the plaque assay was reduced 1.07 log in silenced HepG2 cells