Differential expression of microRNAs in plasma of patients with colorectal cancer: A potential marker for colorectal cancer screening

Objective: MicroRNAs (miRNAs) have been shown to offer great potential in the diagnosis of cancer. We investigated whether plasma miRNAs could discriminate between patients with and without colorectal cancer (CRC). Methods: This study was divided into three phases: (1) marker discovery using real-time PCR-based miRNA profiling on plasma, corresponding cancerous and adjacent non-cancerous colonic tissues of five patients with CRC, along with plasma from five healthy individuals as controls; (2) marker selection and validation by real-time quantitative RT-PCR on a small set of plasma; and (3) independent validation on a large set of plasma from 90 patients with CRC, 20 patients with gastric cancer, 20 patients with inflammatory bowel disease (IBD) and 50 healthy controls. Results: Of the panel of 95 miRNAs analysed, five were upregulated both in plasma and tissue samples. All the five miRNAs were validated on the plasma of 25 patients with CRC and 20 healthy controls. Both miR-17-3p and miR-92 were significantly elevated in the patients with CRC (p<0.0005). The plasma levels of these markers were significantly reduced after surgery in 10 patients with CRC (p<0.05). Further validation with an independent set of plasma samples (n=180) indicated that miR-92 differentiates CRC from gastric cancer, IBD and normal subjects. This marker yielded a receiver operating characteristic curve area of 88.5%. At a cut-off of 240 (relative expression in comparison to RNU6B snRNA), the sensitivity was 89% and the specificity was 70% in discriminating CRC from control subjects. Conclusion: MiR-92 is significantly elevated in plasma of patients with CRC and can be a potential non-invasive molecular marker for CRC screening.published_or_final_versio

Autophagy Induction Is a Tor- and Tp53-Independent Cell Survival Response in a Zebrafish Model of Disrupted Ribosome Biogenesis

Ribosome biogenesis underpins cell growth and division. Disruptions in ribosome biogenesis and translation initiation are deleterious to development and underlie a spectrum of diseases known collectively as ribosomopathies. Here, we describe a novel zebrafish mutant, titania (tti(s450)), which harbours a recessive lethal mutation in pwp2h, a gene encoding a protein component of the small subunit processome. The biochemical impacts of this lesion are decreased production of mature 18S rRNA molecules, activation of Tp53, and impaired ribosome biogenesis. In tti(s450), the growth of the endodermal organs, eyes, brain, and craniofacial structures is severely arrested and autophagy is up-regulated, allowing intestinal epithelial cells to evade cell death. Inhibiting autophagy in tti(s450) larvae markedly reduces their lifespan. Somewhat surprisingly, autophagy induction in tti(s450) larvae is independent of the state of the Tor pathway and proceeds unabated in Tp53-mutant larvae. These data demonstrate that autophagy is a survival mechanism invoked in response to ribosomal stress. This response may be of relevance to therapeutic strategies aimed at killing cancer cells by targeting ribosome biogenesis. In certain contexts, these treatments may promote autophagy and contribute to cancer cells evading cell death.This research was funded by the National Health and Medical Research Council of Australia through Project grant 433614 (JKH), Program grant 487922 (JKH), a Senior Research Fellowship (JKH), and a Howard Florey Centenary Fellowship (HV). Operational Infrastructure Support was provided by the Victorian Government, Australia. Additional support was from Australian Research Council grant DP0346823 (GJL); NIH grant DK060322 (DYRS); and CDMRP, Department of Defense, USA W81XWH-10-1-0854 (KCE)

Scale-free static and dynamical correlations in melts of monodisperse and Flory-distributed homopolymers: A review of recent bond-fluctuation model studies

It has been assumed until very recently that all long-range correlations are screened in three-dimensional melts of linear homopolymers on distances beyond the correlation length $\xi$ characterizing the decay of the density fluctuations. Summarizing simulation results obtained by means of a variant of the bond-fluctuation model with finite monomer excluded volume interactions and topology violating local and global Monte Carlo moves, we show that due to an interplay of the chain connectivity and the incompressibility constraint, both static and dynamical correlations arise on distances $r \gg \xi$. These correlations are scale-free and, surprisingly, do not depend explicitly on the compressibility of the solution. Both monodisperse and (essentially) Flory-distributed equilibrium polymers are considered.Comment: 60 pages, 49 figure

Sfrp5 Modulates Both Wnt and BMP Signaling and Regulates Gastrointestinal Organogensis in the Zebrafish, Danio rerio

Sfrp5 belongs to the family of secreted frizzled related proteins (Sfrp), secreted inhibitors of Wingless-MMTV Integration Site (Wnt) signaling, which play an important role in cancer and development. We selected sfrp5 because of its compelling expression profile in the developing endoderm in zebrafish, Danio rerio. In this study, overexpression of sfrp5 in embryos results in defects in both convergent extension (CE) by inhibition of non-canonical Wnt signaling and defects in dorsoventral patterning by inhibition of Tolloid-mediated proteolysis of the BMP inhibitor Chordin. From 25 hours post fertilization (hpf) to 3 days post fertilization (dpf), both overexpression and knockdown of Sfrp5 decrease the size of the endoderm, significantly reducing liver cell number. At 3 dpf, insulin-positive endodermal cells fail to coalesce into a single pancreatic islet. We show that Sfrp5 inhibits both canonical and non-canonical Wnt signaling during embryonic and endodermal development, resulting in endodermal abnormalities. © 2013 Stuckenholz et al

Simulations of an observed elevated mesoscale convective system over southern England during CSIP IOP 3

Simulations of an elevated mesoscale convective system (MCS) observed over southern England during the Convective Storm Initiation Project (CSIP) provide the first detailed modelling study of a case of elevated convection occurring in the UK. The study shows that many factors can influence the maintenance of elevated deep convection, from large-scale flow through to surface heating processes and diabatic cooling within the convective system. It is also shown that interactions and feedback mechanisms between a stable layer and the storm can act to maintain deep convection. The simulation successfully reproduced an elevated MCS above a low-level stable undercurrent, with a wave in the undercurrent linked to a rear-inflow jet (RIJ). Convection was fed from an elevated (840hPa) source layer with CAPE of about 350Jkg-1. The undercurrent in the simulation was approximately 1km deep, about half that observed. Unlike the observed MCS, a transition from elevated to surface-based convection occurred in the simulation due to the combined effects of a pre-existing large-scale θe gradient, advection and surface heating causing the system to encounter increasingly unstable low-level air and a shallower stable layer that was more susceptible to penetration by downdraughts. The transition to surface-based convection was accompanied by the development of cold-pool outflow and an increase in system velocity from about 6 to 10ms-1. Diabatic cooling from microphysical processes in the simulation enhanced the undercurrent and strengthened the RIJ. This strengthened the wave in the undercurrent and led to more extensive convection. The existence of a positive feedback process between the convection, RIJ and stable layer is discussed. Uncertainty in the synoptic scale generating errors in the undercurrent is shown to be a major source of error for convective-scale forecasts

Imitation of hand and tool actions is effector-independent

Following the theoretical notion that tools often extend one’s body, in the present study, we investigated whether imitation of hand or tool actions is modulated by effector-specific information. Subjects performed grasping actions toward an object with either a handheld tool or their right hand. Actions were initiated in response to pictures representing a grip at an object that could be congruent or incongruent with the required action (grip-type congruency). Importantly, actions could be cued by means of a tool cue, a hand cue, and a symbolic cue (effector-type congruency). For both hand and tool actions, an action congruency effect was observed, reflected in faster reaction times if the observed grip type was congruent with the required movement. However, neither hand actions nor tool actions were differentially affected by the effector represented in the picture (i.e., when performing a tool action, the action congruency effect was similar for tool cues and hand cues). This finding suggests that imitation of hand and tool actions is effector-independent and thereby supports generalist rather than specialist theories of imitation

Purification and Characterization of Enterovirus 71 Viral Particles Produced from Vero Cells Grown in a Serum-Free Microcarrier Bioreactor System

[[abstract]]Background: Enterovirus 71 (EV71) infections manifest most commonly as a childhood exanthema known as hand-foot-and-mouth disease (HFMD) and can cause neurological disease during acute infection. Principal Finding: In this study, we describe the production, purification and characterization of EV71 virus produced from Vero cells grown in a five-liter serum-free bioreactor system containing 5 g/L Cytodex 1 microcarrier. The viral titer was >106 TCID50/mL by 6 days post infection when a MOI of 10?5 was used at the initial infection. Two EV71 virus fractions were separated and detected when the harvested EV71 virus concentrate was purified by sucrose gradient zonal ultracentrifugation. The EV71 viral particles detected in the 24–28% sucrose fractions had an icosahedral structure 30–31 nm in diameter and had low viral infectivity and RNA content. Three major viral proteins (VP0, VP1 and VP3) were observed by SDS-PAGE. The EV71 viral particles detected in the fractions containing 35–38% sucrose were 33–35 nm in size, had high viral infectivity and RNA content, and were composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. The two virus fractions were formalin-inactivated and induced high virus neutralizing antibody responses in mouse immunogenicity studies. Both mouse antisera recognized the immunodominant linear neutralization epitope of VP1 (residues 211–225). Conclusion:These results provide important information for cell-based EV71 vaccine development, particularly for the preparation of working standards for viral antigen quantification

Pilot Scale Production of Highly Efficacious and Stable Enterovirus 71 Vaccine Candidates

BACKGROUND: Enterovirus 71 (EV71) has caused several epidemics of hand, foot and mouth diseases (HFMD) in Asia and now is being recognized as an important neurotropic virus. Effective medications and prophylactic vaccine against EV71 infection are urgently needed. Based on the success of inactivated poliovirus vaccine, a prototype chemically inactivated EV71 vaccine candidate has been developed and currently in human phase 1 clinical trial. PRINCIPAL FINDING: In this report, we present the development of a serum-free cell-based EV71 vaccine. The optimization at each step of the manufacturing process was investigated, characterized and quantified. In the up-stream process development, different commercially available cell culture media either containing serum or serum-free was screened for cell growth and virus yield using the roller-bottle technology. VP-SFM serum-free medium was selected based on the Vero cell growth profile and EV71 virus production. After the up-stream processes (virus harvest, diafiltration and concentration), a combination of gel-filtration liquid chromatography and/or sucrose-gradient ultracentrifugation down-stream purification processes were investigated at a pilot scale of 40 liters each. Although the combination of chromatography and sucrose-gradient ultracentrifugation produced extremely pure EV71 infectious virus particles, the overall yield of vaccine was 7-10% as determined by a VP2-based quantitative ELISA. Using chromatography as the downstream purification, the virus yield was 30-43%. To retain the integrity of virus neutralization epitopes and the stability of the vaccine product, the best virus inactivation was found to be 0.025% formalin-treatment at 37 °C for 3 to 6 days. Furthermore, the formalin-inactivated virion vaccine candidate was found to be stable for >18 months at 4 °C and a microgram of viral proteins formulated with alum adjuvant could induce strong virus-neutralizing antibody responses in mice, rats, rabbits, and non-human primates. CONCLUSION: These results provide valuable information supporting the current cell-based serum-free EV71 vaccine candidate going into human Phase I clinical trials